Pancreatic cancer targets and uses thereof

ABSTRACT

The present invention provides a method for diagnosing and detecting diseases associated with pancreas. The present invention provides one or more proteins or fragments thereof, peptides or nucleic acid molecules differentially expressed in pancreatic diseases (PCAT) and antibodies binds to PCAT. The present invention provides that PCAT is used as targets for screening agents that modulates the PCAT activities. Further, the present invention provides methods for treating diseases associated with pancreas.

This application claims priority under 35 USC .sctn. 119(e) to U.S.Provisional Application No. 60/493,369, filed Aug. 8, 2003; U.S.Provisional Application No. 60/512,690, filed Oct. 21, 2003; U.S.Provisional Application No. 60/519,832, filed Nov. 14, 2003; and U.S.Provisional Application No. 60/552,390, filed Mar. 12, 2004.

FIELD OF THE INVENTION

This invention relates to the fields of molecular biology and oncology.Specifically, the invention provides a molecular marker and atherapeutic agent for use in the diagnosis and treatment of cancers.

BACKGROUND OF THE INVENTION

Cancer currently constitutes the second most common cause of death inthe United States. Carcinomas of the pancreas are the eighth mostprevalent form of cancer and fourth among the most common causes ofcancer deaths in this country.

The prognosis for pancreatic carcinoma is, at present, very poor, itdisplays the lowest five-year survival rate among all cancers. Suchprognosis results primarily from delayed diagnosis, due in part to thefact that the early symptoms are shared with other more common abdominalailments. Despite the advances in diagnostic imaging methods likeultrasonography (US), endoscopic ultrasonography (EUS), dualphase spiralcomputer tomography (CT), magnetic resonance imaging (MRT), endoscopicretrograde cholangiopancreatography (ERCP) and transcutaneous orEUS-guided fine-needle aspiration (FNA), distinguishing pancreaticcarcinoma from benign pancreatic diseases, especially chronicpancreatitis, is difficult because of the similarities in radiologicaland imaging features and the lack of specific clinical symptoms forpancreatic carcinoma.

Substantial efforts have been directed to developing tools useful forearly diagnosis of pancreatic carcinomas. Nonetheless, a definitivediagnosis is often dependent on exploratory surgery which is inevitablyperformed after the disease has advanced past the point when earlytreatment may be effected.

One promising method for early diagnosis of various forms of cancer isthe identification of specific biochemical moieties, termed targets,expressed differentially in the cancerous cells. The targets are eithercell suface proteins or cytosolic proteins. Antibodies which willspecifically recognize and bind to the targets in the cancerous cellspotentially provide powerful tools for the diagnosis and treatment ofthe particular malignancy.

SUMMARY OF THE INVENTION

The present invention is based on the identification of certain cellsurface proteins or cytosolic proteins that are differentially expressedin pancreatic disease. A malignant cell often differs from a normal cellby a differential expression of one or more proteins. Thesedifferentially expressed proteins, and the fragments thereof, areimportant markers for the diagnosis of pancreatic disease, as well asother cancers. The differentially expressed proteins of the presentinvention and the nucleic acids encoding said proteins and the fragmentsof said proteins are referred to herein as pancreatic cancer associatedtarget, PCAT proteins or PCAT nucleic acids or PCAT peptides,respectively.

The present invention provides peptides and protein differentiallyexpressed in pancreatic diseases (hereinafter PCAT), particularlypancreatic cancer. Based on the site of protein localization, e.g.,surface or cytosolic, and protein characterization, e.g. receptor orenzyme, specific uses of these PCATs are provided. Some of the PCATs ofthe present invention serve as targets for one or more classes oftheraputic agents, while others may be suitable for antibodytheraputics.

Accordingly, the present invention provides a method for diagnosing ordetecting a pancreatic disease in a subject comprising: determining thelevel of one or more PCAT proteins, or any fragment(s) thereof, in atest sample from said subject, wherein said PCAT protein comprises asequence selected from a group consisting of SEQ ID NOS: 1-288; whereina differential level of said PCAT protein(s) or fragment(s) in saidsample relative to the level of said protein(s) or fragment(s) in a testsample from a healthy subject, or the level established for a healthysubject, is indicative of pancreatic disease.

The present invention also provides a method for detecting a pancreaticdisease in a subject comprising: determining the level of one or morePCAT peptide(s) comprising a peptide sequence selected from a groupconsisting of SEQ ID NOS: 703-873 in a test sample from said subject,wherein a differential level of said PCAT peptide(s) in said sample tothe level of said PCAT peptide(s) in a test sample from a healthysubject, or the level of said PCAT peptide(s) established for a healthysubject, is indicative of the pancreatic disease.

The present invention further provides a method for detecting apancreatic disease in a subject comprising: determining the level of oneor more PCAT nucleic acid(s), or any fragment(s) thereof, in a testsample from said subject, wherein said PCAT nucleic acid(s) encode aPCAT protein sequence selected from a group consisting of SEQ ID NOS:1-288; wherein a differential level of said PCAT nucleic acids orfragment(s) in said sample relative to the level of said protein(s) orfragment(s) in a test sample from a healthy subject, or the levelestablished for a healthy subject, is indicative of the pancreaticdisease.

The invention also provides methods for detecting the PCAT peptides,gene or mRNA in a test sample for use in diagnosing the presence,absence or progression of a disease. The test sample includes but is notlimited to a biological sample such as tissue, blood, serum orbiological fluid.

The present invention further provides a purified antibody that bindsspecifically to a protein molecule, or any fragment thereof, selectedfrom a group consisting of SEQ ID NOS: 1-288.

The present invention further provides a composition comprising anantibody that binds to a protein selected from a group consisting of SEQID NOS: 1-288, and an acceptable carrier.

The present invention further provides a method for treating apancreatic disease, comprising administering to a patient in need ofsaid treatment a therapeutically effective amount of one or moreantibody(ies) of this invention.

The present invention further provides a method for treating apancreatic disease comprising (i) identifying a subject havingpancreatic disease and (ii) administering to a said patient atherapeutically effective amount of one or more antibody(ies) of thisinvention.

The present invention further provides a method to screen for agentsthat modulate PCAT protein activity, comprising the steps of (i)contacting a test agent with a PCAT protein and (ii) assaying for PCATprotein activity, wherein a change in said activity in the presence ofsaid agent relative to PCAT protein activity in the absence of saidagent indicates said agent modulates said PCAT protein activity.

The present invention further provides a method to screen for agentsthat bind to PCAT proteins, comprising the steps of (i) contacting atest agent with a PCAT protein and (ii) measuring the level of bindingof agent to said PCAT protein.

The invention also provides diagnostic methods for human disease, inparticular for pancreatic cancer, its metastatic stage, and therapeuticpotential.

The present invention further provides diagnostic method forepithelial-cell related cancers. In particular, pancreas, lung, colon,prostate, ovarian, breast, bladder renal, hepatocellular, pharyngeal,and gastric cancers.

The invention also provides a method for monitoring the diseaseprogression and the treatment progress.

The invention further provide a method of diagnosis by an array, whereinthe array is immobilized with two or more PCAT proteins, peptides ornucleic acid molecules. The proteins, peptides or nucleic acid moleculesinclude but are not limited to the SEQ ID NOS: 1-873.

The invention also provides monoclonal or polyclonal antibodies andcomposition thereof reactive with antigentic portion of PCAT protein,peptides or fragments thereof in a form for use in pancreatic diseasesdiagnosis.

The invention further provides an immunogenic antibody for treatingpancreatic disease or diseases associated with pancreatic diseases,preferably pancreatic cancer.

The present invention provides a method for screening agents thatmodulate PCAT activity, comprising the steps of (a) contacting a samplecomprising a PCAT with an agent; and (b) assaying for PCAT activity,wherein a change in said PCAT activity in the presence of said agentrelative to PCAT activity in the absence of said compound indicates saidagent modulates PCAT. The agents include but are not limted to protein,peptide, antibody, nucleic acid such as antisense RNA, RNAi fragments,small molecules.

The present invention further provides a method for treating pancreaticdiseases, comprising: administering to a patient one or more agents in atherapeutically effective amount to treat pancreatic diseases.

The present invention provides a method for treating pancreaticdiseases, comprising: identifying a subject having pancreatic diseases;and administering to a patient to one or more antibodies in atherapeutically effective amount to treat pancreatic diseases.

The present invention further provide method for diagnosis and treatmentfor pancreatic cancer.

The present invention further provides therapeutic potential forepithelial-cell related cancers. In particular pancreas, lung, colon,prostate, ovarian, breast, bladder renal, hepatocellular, pharyngeal andgastric cancers.

Description of the Files Contained on the CD-R Named CL001538CDR

The CD-R named CL001538CDR contains the following three text (ASCII)files:

1) File 1538 SEQLIST txt provides the Sequence Listing. The SequenceListing provides the protein sequences (SEQ ID NOS: 1-288); transcriptsequences (SEQ ID NOS: 289-702) and peptide sequences (SEQ ID NOS:703-873) as shown in Table 2. File SEQLIST_(—)1538.txt is 3,230 KB insize.2) File 1538 prov_Applera_Proteomic_IP_table1 is 185 KB in size.3) File 1538 prov_Applera_Proteomic_IP_table2 is 1984 KB in size.

The material contained on the CD-R labeled CL001538 CDR is herebyincorporated by reference pursuant to 37 CFR 1.77(b)(4).

Description of Table 1 and Table 2

Table 1 (provided on the CD-R) discloses the peptides which correspondto the protein, the pancreatic cancer cell lines, the expressioninformation, the ratio compare to the control sample. The expression isbased on measuring the level of the peptides. Numerical representationof overexpression is indicated by more than two, whereas numericalrepresentation of underexpression is indicated by less than 0.5.Overexpressed singleton indicates that the peptide peak in diseasedsample was detected and there was no peak detected in control samples.Underexpressed singleton indicates that the peptide peak was detected inthe control sample and there was no peak in the diseased sample.

Table 2 (provided on the CD-R) discloses the PCAT proteins, transcripts,and peptides sequences that correlated to the Table 1.

The transcript/protein information includes:

a protein number (1 through 288)

a Celera protein internal identification number for the protein encodedby the Celera transcript (hCP and/or UID)

a public protein accession number (Genbank e.g., RefSeq NP number,Swiss-prot, or Derwent) for the protein

an art-known gene/protein name

a Celera transcript internal identification number (hCT and/or UID)

a public transcript accession number (Genbank e.g., RefSeq NM number, orDerwent)

a Celera hCG and UID internal identification numbers for the gene

an art-known gene symbol

Celera genomic axis position (indicating start nucleotide position-stopnucleotide position)

the chromosome number of the chromosome on which the gene is located

an OMIM (Online Mendelian Inheritance in Man; Johns HopkinsUniversity/NCBI) public reference number for obtaining furtherinformation regarding the medical significance of each gene

alternative gene/protein name(s) and/or symbol(s) in the OMIM entry

DESCRIPTION OF FIGURES

FIG. 1. Tissue Factor expression in various tumor types byimmunohistochemistry.

FIG. 2. Kunitz-type 1 protease inhibitor expression in various tumortypes by immunohistochemistry.

FIG. 3. Mucin 4 expression in various tumor types byimmunohistochemistry.

FIG. 4. Kunitz-type 1 protease inhibitor mRNA expression in pancreaticcell lines. Kunitz-type 1 protease inhibitor mRNA expression wasmeasured across the pancreatic cell line panel and calculated as folddifference from the reference cell line Hs766t. mRNA expression wasfound to be over-expressed in several of the cell lines relative toHs766t, including the SU.86.86 cell line consistent ICAT data collectedby mass spectrometry.

FIG. 5. Caspase 3/7 Analysis of Mucin 4 (PA-116) knockdown. Mucin siRNAinduced apoptosis of ASPC-1 cells 3 days after transfection, and levelof apoptosis was dose-dependent on muscin siRNA (n=3). The level ofapoptosis on day 3 was similar to that given by TRAIL+ actinomycin Dpositive control.

FIG. 6. Knockdown effects on MTS proliferation. Mucin 4 siRNA inhibitedthe proliferation of ASPC-1 cells (n=3). The level of inhibition wasshown to be dose-dependent on the concentration of Mucin 4 siRNA (n=3)

FIG. 7. Viability test on Mucin 4 (PA-116) siRNA after transfection. Theresult shows that Mucin siRNA can inhibit about 50% of viable ASPC-1cells 3 days after transfection (n=2)

FIG. 8. Analysis of individual Mucin 4 (PA-116) siRNA vs. SMARTPool. Theresult shows that both SMARTPool Mucin 4 siRNA and Mucin 4 duplexI siRNAcan induce an inhibition of proliferation and an increase in apoptosis 3days after transfection

FIG. 9. Morphology of ASPC-1 Pancreatic Cancer Cells with Mucin 4 SiRNA

FIG. 10. Target protein expression was measured in whole cell extractsprepared from the pancreatic cell line panel. Polyclonal antibodiesraised in rabbits (Green Mountain Antibodies) were used to detect Gs3786or Retinol Dehydrogenase Type II homolog. Targets were found to beover-expressed in several cell lines relative to the reference cell lineHs766t.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

General Description

While the broadest definition of this invention is set forth in theSummary of the Invention, certain nucleic acids, peptides or proteinsare preferred. For example a preferred method for detecting a pancreaticdisease by determining the level of one or more PCAT protein(s) or anyfragment(s) thereof is wherein the level of PCAT protein(s) aredetermined by contacting one or more antibody(ies) that specificallybind to the antigenic regions of the PCAT protein(s). Further preferredis a method wherein the level of two or more proteins are determined,more preferred wherein the level of four or more proteins are determinedand most preferred wherein the level of eight or more proteins aredetermined.

A preferred method for detecting a pancreatic disease by determining thelevel of one or more PCAT peptide(s) is wherein the level of PCATpeptides(s) are determined by contacting one or more antibody(ies) thatspecifically bind to the antigenic regions of the PCAT peptide(s).Further preferred is a method wherein the level of five or more peptidesare determined, more preferred wherein the level of ten or more peptidesare determined and most preferred wherein the level of fifteen or morepeptides are determined.

A preferred method for detecting a pancreatic disease by determining thelevel of one or more PCAT nucleic acid(s) is wherein the level of saidPCAT nucleic acid(s) is determined by contacting one or more probes thatspecifically hybridize to said nucleic acid(s). Further preferred is amethod wherein the level of two or more nucleic acids are determined,more preferred wherein the level of four or more nucleic acids aredetermined and most preferred wherein the level of eight or more nucleicacids are determined.

The methods for detecting a pancreatic disease provided by the presentinvention may be used for diagnosing the presence of disease in apatent, monitoring the presence of pancreatic disease in patientsundergoing treatment and testing for the reoccurrence of pancreaticdisease in patients that were successfully treated for the pancreaticdisease; preferably wherein the pancreatic disease is pancreatic cancer.The test sample may be, but is not limited to, a biological sample suchas tissue, blood, serum or biological fluid.

The present invention is based on the discovery of protein(s) andpeptide(s) that are differentially expressed in pancreatic diseasesamples, for example, cancer sample versus normal pancreatic samples.These proteins and peptide, and the encoding nucleic acid molecules areassociated with pancreatic diseases, hereinafter the PCAT proteins,peptides or nucleic acids.

The discovery of disease specific target proteins is base on discoveriesmade using proteomics techniques. The method uses on MALDI-TOF TOF LC/MSanalyses platform to generate protein expression profiles frompancreatic disease tissues or cell lines in an effort to discover andidentify novel molecules associated with the disease.

Based on these discoveries, the present invention provides proteins,peptides, nucleic acids that are differential in pancreatic diseases, aswell as antibodies binds to the proteins or peptides. The presentinvention also provides methods for detection, monitoring, diagnosis,prognosis, preventive and treatment of pancreatic diseases. The presentinvention provides a detection reagent, markers for pancreatic diseasesat various stages, comprises PCAT sequences isolated from humanpancreatic disease tissue, sera, cell lines, blood or biological fluids.

The present invention provides a method for treating pancreatic diseasestargeting at PCAT. The treatment includes administration of atherapeutically effective amount of composition comprise, but not limitto, an antibody, an immunogentic peptide which induces T cell response,a small molecule, a protein or a nucleic acid molecule. The compositionfurther comprises an agonist or antigonist to PCAT. A “Panceraticdisease” can include pancreatic cancer, pancreatic tumor (exocrine orendocrine), pancreatic cysts, acute pancreatitis, chronic pancreatitis,diabetes (type I and II) as well as pancreatic trauma, preferablypancreatic cancer.

The present invention may further provide a diagnostic or therapeuticpotential for epithelial-cell related cancers, which include but are notlimited to pancreas, lung, colon, prostate, ovarian, breast, bladderrenal, hepatocellular, pharyngeal and gastric cancers.

The present invention further provides the target for screening an agentfor PCAT, wherein the agent is compounds of small molecules, proteins,peptides, nucleic acids, antibodies or other agonists or antigonists.

Specifically, the following targets are selected for targetingpurpose:Tissue Factor, GS3786, Na/K ATPase Beta-3, Kunitz Typeinhibitor-2 (HAI-2), CD46, CD166, Neutral Amino Acid Transporter(ASCT2), Transglutaminase-2 (TGM2), Kunitz Inhibitor-1, ErbB3, Mucin 4,Decay Accelerating Factor (DAF). These factors are associated withpancreatic cancer and are the potential target for treating pancreaticcancer.

Tissue factor is known for its function for activation of fVII resultingin genenration of tumor growth promoting factors. fVIIa is known to bindTF and may stimulate mitogenic signaling and inhibit apoptosis of tumorcells.

Mucin 4 is a transmembrane mucin composed of two subunits (alpha andbeta) generated from proteolytic cleavage (beta is transmembrane, alphais extracellular). Mucin 4 and erbB2 is localized to the apical membraneand stimulates differentiation of polarized epithelium. Expression ofneuregulin[NRG] by stromal elements enables erbB2/erbB3 complexformation which stimulates proliferation. Loss of polarization ofepithelial cell allows Mucin 4/erbB2 and erbB3 complex formation, whichpotentiates proliferative signaling. ErbB3 is unique among the ErbBreceptor family in that it possesses little or no intrinsic tyrosinekinase activity. When ErbB3 is co-expressed with ErbB2, an activesignaling complex is formed and antidies directded against ErbB2 arecapable of disrupting this complex. Monoclonal antibody against ErbB3has an agonistic effect on the anchorage-independent growth of celllines expressing this receptor (Rajkumar et al., British J. Cancer,70(3): 459-465 (1994)).

Kunitz Type Protease Inhibitor-1, also called HAI-1, Serine proteaseinhibitors or serpins. HAI is associate with hepatocyte growth factorHGF/scatter factor/SF which is thought to play an important role in theregeneration of injured gastrointestinal mucosa by promoting theproliferation and migration of epithelial cells. HGF is activated byHGFA which is activated locally by thrombin. HAI-1 is upregulated andinhibits and sequesters HGFA at the site of injury, thereby ensuringpericellular activation of HGF through upregulation of injury associatedproteases. HGFA is released from HAI-1 upon cleavage with cell surfacemetalloprotease.

Neutral Amino Acid Transporter (ASCT2) is a member of ASC/BO amino acidtransporter and it functions as Na+ dependent glutamine transport.

Decay accelerating factor (CD55)'s primary function is to inactivate theC3 convertases of the complement cascade by dissociating them into theirconstituent proteins. It is expressed on all serum exposed cells andprovides protection from complement activated proteases.

PCAT Peptide/Proteins and Peptides

The present invention provides isolated PCAT peptide and proteinmolecules that consisting of, consisting essentially of, or comprisingthe amino acid sequences of the PCAT peptides and proteins disclosed inthe Tables 1 and 2, (encoded by the nucleic acid molecule shown in Table2), as well as all obvious variants of these peptides that are withinthe art to make and use. Some of these variants are described in detailbelow.

In one embodiment PCAT peptides include, but are not limited to, theamino acid sequence of SEQ ID NOS: 703-873 and variants thereof. A PCATprotein includes, but is not limited to, the amino acid sequence of SEQID NOS: 1-288 and variants thereof. PACT proteins may be differentiallyexpressed in pancreatic cell line, blood, tissue, serum or body fluids.

The peptide or protein or fragement thereof, to which the inventionpertains, however, are not to be construed as encompassing peptide,protein or fragment that may be disclosed publicly prior to the presentinvention.

The PCAT proteins and peptides of the present invention can be purifiedto homogeneity or other degrees of purity. The level of purificationwill be based on the intended use. The critical feature is that thepreparation allows for the desired function of the peptide, even if inthe presence of considerable amounts of other components (the featuresof an isolated nucleic acid molecule is discussed below).

As used herein, a “peptide” is defined as amino acid sequences between5-20 amino acids derived from PCAT proteins such as SEQ ID NOS: 1-288 orvariants thereof. The peptide differentially expressed in eitherpancreatic diseases cell line, blood, tissue, serum or body fluids. Inone embodiment peptides include, but are not limited to, the amino acidsequence of SEQ ID NOS: 703-873, or variants thereof.

As used herein, a “protein” is full-length protein differentiallyexpressed in pancreatic diseases cell line, tissue, blood, serum or bodyfluids. A protein includes, but is not limited to, the amino acidsequence of SEQ ID NOS: 1-288.

A peptide is said to be “isolated” or “purified” when it issubstantially free of cellular material or free of chemical precursorsor other chemicals. The peptides of the present invention can bepurified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule are discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thePCAT peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 50% chemical precursors or otherchemicals.

The isolated PCAT proteins and peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd.ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(2001). Experimental data as provided in Table 1 indicates expression inhumans pancreatic cell lines. For example, a nucleic acid moleculeencoding the PCAT protein or peptide is cloned into an expressionvector, the expression vector introduced into a host cell and theprotein expressed in the host cell. The protein or peptide can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques. Many of these techniques aredescribed in detail below.

A PCAT peptide or protein can be attached to heterologous sequences toform chimeric or fusion proteins. Such Schimeric and fusion proteinscomprise a peptide operatively linked to a heterologous protein havingan amino acid sequence not substantially homologous to the peptide.“Operatively linked” indicates that the peptide and the heterologousprotein are fused in-frame. The heterologbus protein can be fused to theN-terminus or C-terminus of the peptide.

In some uses, the fusion protein does not affect the activity of thepeptide or protein per se. For example, the fusion protein can include,but is not limited to, fusion proteins, for example beta-galactosidasefusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged,HI-tagged and Ig fusions. Such fusion proteins, particularly poly-Hisfusions, can facilitate the purification of recombinant PCAT proteins orpeptides. In certain host cells (e.g., mammalian host cells), expressionand/or secretion of a protein can be increased by using a heterologoussignal sequence.

A chimeric or fusion PCAT protein or peptide can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A PCAT-encoding nucleic acid can becloned into such an expression vector such that the fusion moiety islinked in-frame to the PCAT protein or peptide.

As mentioned above, the PCAT peptide or the PCAT protein has obviousvariants of the amino acid sequence, such as naturally occurring matureforms of the PCAT, allelic/sequence variants of the PCAT, non-naturallyoccurring recombinantly derived variants of the PCAT, and orthologs andparalogs of the PCAT proteins or peptides. Such variants can readily begenerated using art-known techniques in the fields of recombinantnucleic acid technology and protein biochemistry.

It is understood, however, that PCAT and variants exclude any amino acidsequences disclosed prior to the invention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the PCAT peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48): 444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package, usingeither a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16,14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. Inyet another preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (Devereux, J., et al., Nucleic Acids Res. 12(1): 387(1984)), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In anotherembodiment, the percent identity between two amino acid or nucleotidesequences is determined using the algorithm of E. Myers and W. Miller(CABIOS, 4: 11-17 (1989)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol. 215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17): 3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thePCAT peptides of the present invention as well as being encoded by thesame genetic locus as the PCAT peptide provided herein (See Table 2).

Allelic variants of a PCAT peptide can readily be identified as being ahuman protein having a high degree (significant) of sequencehomology/identity to at least a portion of the PCAT peptide as well asbeing encoded by the same genetic locus as the PCAT peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in Table 2, such as the genomic sequence mapped tothe reference human. As used herein, two proteins (or a region of theproteins) have significant homology when the amino acid sequences aretypically at least about 70-80%, 80-90%, and more typically at leastabout 90-95% or more homologous. A significantly homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence that will hybridize to a PCAT peptide encodingnucleic acid molecule under stringent conditions as more fully describedbelow.

Paralogs of a PCAT peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the PCAT peptide, as being encoded by a gene from humans, and ashaving similar activity or function. Two proteins will typically beconsidered paralogs when the amino acid sequences are typically at leastabout 60% or greater, and more typically at least about 70% or greaterhomology through a given region or domain. Such paralogs will be encodedby a nucleic acid sequence that will hybridize to a PCAT peptideencoding nucleic acid molecule under moderate to stringent conditions asmore fully described below.

Orthologs of a PCAT peptide can readily be identified as having somedegree of significant sequence homology/identity to at least a portionof the PCAT peptide as well as being encoded by a gene from anotherorganism. Preferred orthologs will be isolated from mammals, preferablyprimates, for the development of human therapeutic targets and agents.Such orthologs will be encoded by a nucleic acid sequence that willhybridize to a PCAT peptide encoding nucleic acid molecule undermoderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

Non-naturally occurring variants of the PCAT peptides of the presentinvention can readily be generated using recombinant techniques. Suchvariants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the PCAT peptide. Forexample, one class of substitutions is conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a PCAT peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247: 1306-1310 (1990).

Variant PCAT peptides can be fully functional or can lack function inone or more activities, e.g. ability to bind substrate, ability tophosphorylate substrate, ability to mediate signaling, etc. Fullyfunctional variants typically contain only conservative variation orvariation in non-critical residues or in non-critical regions.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244: 1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity such as PCAT activity or in assays such as an invitro proliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224: 899-904 (1992); de Vos et al.Science 255: 306-312 (1992)).

The present invention further provides fragments of the PCATs, inaddition to proteins and peptides that comprise and consist of suchfragments, particularly those comprising the residues identified inTables 1 and 2. As used herein, a fragment comprises at least 8, 10, 12,14, 16, 18, 20 or more contiguous amino acid residues from a PCAT. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the PCAT or could be chosen for the abilityto perform a function, e.g. bind a substrate or act as an immunogen.Particularly important fragments are biologically active fragments,peptides that are, for example, about 8 or more amino acids in length.Such fragments will typically comprise a domain or motif of the PCAT,e.g., active site, a transmembrane domain or a substrate-binding domain.Further, possible fragments include, but are not limited to, domain ormotif containing fragments, soluble peptide fragments, and fragmentscontaining immunogenic structures. Predicted domains and functionalsites are readily identifiable by computer programs well known andreadily available to those of skill in the art (e.g., PROSITE analysis).

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in PCATs are described inbasic texts, detailed monographs, and the research literature, and theyare well known to those of skill in the art.

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663: 48-62(1992)).

Accordingly, the PCATs of the present invention also encompassderivatives or analogs in which a substituted amino acid residue is notone encoded by the genetic code, in which a substituent group isincluded, in which the mature PCAT is fused with another compound, suchas a compound to increase the half-life of the PCAT (for example,polyethylene glycol), or in which the additional amino acids are fusedto the mature PCAT, such as a leader or secretory sequence or a sequencefor purification of the mature PCAT or a pro-protein sequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the functional information provided in theTables; to raise antibodies or to elicit another immune response; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a PCAT-effectorprotein interaction or PCAT-ligand interaction), the protein can be usedto identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 3rd ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,2001, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, PCATs isolated from humans and theirhuman/mammalian orthologs serve as, targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the PCAT. Experimental data as provided inTable 1 indicate that the PCATs of the present invention are expressedat differential level in various pancreatic cell lines, for example SEQID NOS: 1-119 are overexpressed in all tested cell lines, whereas otherproteins or peptides are underexpressed in selected cell lines. In oneexample, protein (SEQ ID NO: 120) or peptide (SEQ ID NO: 761) isoverexpressed in one cell line (HPAC), yet underexpressed in anothercell line (Su.86.86). A large percentage of pharmaceutical agents arebeing developed that modulate the activity of PCAT proteins,particularly members of the PCAT subfamily (see Background of theInvention). The structural and functional information provided in theBackground and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in Table 1. Experimental data asprovided in Table 1 indicates expression in humans pancreatic celllines. Such uses can readily be determined using the informationprovided herein, that which is known in the art, and routineexperimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to PCATs that are related to members ofthe PCAT subfamily. Such assays involve any of the known PCAT functionsor activities or properties useful for diagnosis and treatment ofPCAT-related conditions that are specific for the subfamily of PCATsthat the one of the present invention belongs to, particularly in cellsand tissues that express the PCAT. Experimental data as provided inTable 1 indicate that the PCATs of the present invention are expressedat differential level in various pancreatic cell lines, for example SEQID NOS: 1-119 are overexpressed in all tested cell lines, whereas otherproteins or peptides are underexpressed in selected cell lines. In oneexample, protein (SEQ ID NO: 120) or peptide (SEQ ID NO: 761) isoverexpressed in one cell line (HPAC), yet underexpressed in anothercell line (Su.86.86).

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the PCAT, as a biopsy orexpanded in cell culture. Experimental data as provided in Table 1indicates expression in human pancreatic cell lines. In an alternateembodiment, cell-based assays involve recombinant host cells expressingthe PCAT protein.

The polypeptides can be used to identify compounds or agents thatmodulate PCAT activity of the protein in its natural state or an alteredform that causes a specific disease or pathology associated with thePCAT. Both the PCATs of the present invention and appropriate variantsand fragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the PCAT. These compounds can befurther screened against a functional PCAT to determine the effect ofthe compound on the PCAT activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the PCAT to a desired degree.

Further, the proteins of the present invention can be used to screen acompound or an agent for the ability to stimulate or inhibit interactionbetween the PCAT protein and a molecule that normally interacts with thePCAT protein, e.g. a substrate or or an extracellular binding ligand ora component of the signal pathway that the PCAT protein normallyinteracts (for example, a cytosolic signal proein or another PCAT). Suchassays typically include the steps of combining the PCAT protein with acandidate compound under conditions that allow the PCAT protein, orfragment, to interact with the target molecule, and to detect theformation of a complex between the protein and the target or to detectthe biochemical consequence of the interaction with the PCAT protein andthe target, such as any of the associated effects of signal transductionsuch as protein phosphorylation, cAMP turnover, and adenylate cyclaseactivation, etc.

Candidate compounds or agents include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354: 82-84(1991); Houghten et al., Nature 354: 84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g. Songyang etal., Cell 72: 767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)2, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound or agent is a soluble fragment of the PCAT thatcompetes for substrate binding. Other candidate compounds include mutantPCATs or appropriate fragments containing mutations that affect PCATfunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) PCAT activity. The assaystypically involve an assay of events in the signal transduction pathwaythat indicate PCAT activity. Thus, the phosphorylation of a substrate,activation of a protein, a change in the expression of genes that areup- or down-regulated in response to the PCAT protein dependent signalcascade can be assayed.

Any of the biological or biochemical functions mediated by the PCAT canbe used as an endpoint assay. These include all of the biochemical orbiochemical/biological events described herein, in the references citedherein, incorporated by reference for these endpoint assay targets, andother functions known to those of ordinary skill in the art or that canbe readily identified using the information provided in the Tables,particularly Table 1. Specifically, a biological function of a cell ortissues that expresses the PCAT can be assayed. Experimental data asprovided in Table 1 indicate that the PCATs of the present invention areexpressed at differential level in various pancreatic cell lines, forexample SEQ ID NOS: 1-119 are overexpressed in all tested cell lines,whereas other proteins or peptides are underexpressed in selected celllines. In one example, protein (SEQ ID NO: 120) or peptide (SEQ ID NO:761) is overexpressed in one cell line (HPAC), yet underexpressed inanother cell line (Su.86.86).

Binding and/or activating compounds can also be screened by usingchimeric PCAT proteins in which the amino terminal extracellular domain,or parts thereof, the entire transmembrane domain or subregions, such asany of the seven transmembrane segments or any of the intracellular orextracellular loops and the carboxy terminal intracellular domain, orparts thereof, can be replaced by heterologous domains or subregions.For example, a substrate-binding region can be used that interacts witha different substrate then that which is recognized by the native PCAT.Accordingly, a different set of signal transduction components isavailable as an end-point assay for activation. This allows for assaysto be performed in other than the specific host cell from which the PCATis derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the PCAT (e.g. binding partners and/or ligands). Thus, a compoundis exposed to a PCAT polypeptide under conditions that allow thecompound to bind or to otherwise interact with the polypeptide. SolublePCAT polypeptide is also added to the mixture. If the test compoundinteracts with the soluble PCAT polypeptide, it decreases the amount ofcomplex formed or activity from the PCAT. This type of assay isparticularly useful in cases in which compounds are sought that interactwith specific regions of the PCAT. Thus, the soluble polypeptide thatcompetes with the target PCAT region is designed to contain peptidesequences corresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the PCAT protein, or fragment, or its target moleculeto facilitate separation of complexes from uncomplexed forms of one orboth of the proteins, as well as to accommodate automation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofPCAT-binding protein found in the bead fraction quantitated from the gelusing standard electrophoretic techniques. For example, either thepolypeptide or its target molecule can be immobilized utilizingconjugation of biotin and streptavidin using techniques well known inthe art. Alternatively, antibodies reactive with the protein but whichdo not interfere with binding of the protein to its target molecule canbe derivatized to the wells of the plate, and the protein trapped in thewells by antibody conjugation. Preparations of a PCAT-binding proteinand a candidate compound are incubated in the PCAT protein-presentingwells and the amount of complex trapped in the well can be quantitated.Methods for detecting such complexes, in addition to those describedabove for the GST-immobilized complexes, include immunodetection ofcomplexes using antibodies reactive with the PCAT protein targetmolecule, or which are reactive with PCAT protein and compete with thetarget molecule, as well as PCAT-linked assays which rely on detectingan enzymatic activity associated with the target molecule.

Agents that modulate one of the PCATs of the present invention can beidentified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

Modulators of PCAT protein activity identified according to these drugscreening assays can be used to treat a subject with a disorder mediatedby the PCAT pathway, by treating cells or tissues that express the PCAT.Experimental data as provided in Table 1 indicates expression in humanpancreatic cell lines. These methods of treatment include the steps ofadministering a modulator of PCAT activity in a pharmaceuticalcomposition to a subject in need of such treatment, the modulator beingidentified as described herein.

In yet another aspect of the invention, the PCAT proteins can be used as“bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72: 223-232; Maduraet al. (1993) J. Biol. Chem. 268: 12046-12054; Bartel et al. (1993)Biotechniques 14: 920-924; Iwabuchi et al. (p993) Oncogene 8: 1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with the PCAT and are involved in PCAT activity. SuchPCAT-binding proteins are also likely to be involved in the propagationof signals by the PCAT proteins or PCAT targets as, for example,downstream elements of a PCAT-mediated signaling pathway. Alternatively,such PCAT-binding proteins are likely to be PCAT inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a PCAT protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences that encode an unidentified protein (“prey” or“sample”) is fused to a gene that codes for the activation domain of theknown transcription factor. If the “bait” and the “prey” proteins areable to interact, in vivo, forming a PCAT-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the PCATprotein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a PCAT-modulating agent, an antisense PCATnucleic acid molecule, an PCAT-RNAi fragment, a PCAT-specific antibody,or a PCAT-binding partner) can be used in an animal or other model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal or other model to determine the mechanism of action ofsuch an agent. Furthermore, this invention pertains to uses of novelagents identified by the above-described screening assays for treatmentsas described herein.

The PCAT proteins of the present invention are also useful to provide atarget for diagnosing a disease or predisposition to disease mediated bythe peptide. Accordingly, the invention provides methods for detectingthe presence, or levels of, the protein (or encoding mRNA) in a cell,tissue, or organism. Experimental data as provided in Table 1 indicatesexpression in human pancreatic cell lines. The method involvescontacting a biological sample with a compound capable of interactingwith the PCAT protein such that the interaction can be detected. Such anassay can be provided in a single detection format or a multi-detectionformat such as an antibody chip array.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered PCAT activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11): 983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2): 254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes affects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the PCAT protein in which one ormore of the PCAT functions in one population are different from those inanother population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and PCAT activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in Table 1 indicates expression in humanpancreatic cell-lines. Accordingly, methods for treatment include theuse of the PCAT protein or fragments.Antibodies

The present invention provides antibodies specifically bind to PCATproteins or fragments thereof, peptides, or antigenic portion thereof.

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof as describe above.

The antibody of present invention selectively binds a target PCAT whenit binds the target domain and does not significantly bind to unrelatedproteins. An antibody is still considered to selectively bind a peptideeven if it also binds to other proteins that are not substantiallyhomologous with the target peptide so long as such proteins sharehomology with a fragment or domain of the peptide target of theantibody. In this case, it would be understood that antibody binding tothe peptide is still selective despite some degree of cross-reactivity.

The term “antibody” is used in the broadest sense, and specificallycovers monoclonal antibodies (including full length monoclonalantibodies), polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), humanized antibody and antibody fragments (e.g.,Fab, F(ab′).sub.2 and Fv) so long as they exhibit the desired biologicalactivity. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteinshaving the same structural characteristics. While antibodies exhibitbinding specificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules that lack antigenspecificity.

As used herein, antibodies are usually heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two identical light (L) chains andtwo identical heavy (H) chains. Each light chain is linked to a heavychain by one covalent disulfide bond, while the number of disulfidelinkages varies between the heavy chains of different immunoglobulinisotypes. Each heavy and light chain also has regularly spacedintrachain disulfide bridges. Each heavy chain has at one end a variabledomain (VH) followed by a number of constant domains. Each light chainhas a variable domain at one end (VL) and a constant domain at its otherend. The constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light andheavy chain variable domains. Chothia et al., J. Mol. Biol. 186, 651-63(1985); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82 4592-4596(1985).

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of the environment in which isproduced. Contaminant components of its production environment arematerials that would interfere with diagnostic or therapeutic uses forthe antibody, and may include enzymes, hormones, and other proteinaceousor nonproteinaceous solutes. In preferred embodiments, the antibody willbe purified as measurable by at least three different methods: 1) togreater than 95% by weight of antibody as determined by the Lowrymethod, and most preferably more than 99% by weight; 2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequentator; or 3) tohomogeneity by SDS-PAGE under reducing or non-reducing conditions usingCoomasie blue or, preferably, silver stain. Isolated antibody includesthe antibody in situ within recombinant cells since at least onecomponent of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An “antigenic region” or “antigenic determinant” or an “epitope”includes any protein determinant capable of specific binding to anantibody. This is the site on an antigen to which each distinct antibodymolecule binds. Epitopic determinants usually consist of active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three-dimensional structural characteristics, aswell as charge characteristics.

“Antibody specificity,” is an antibody, which has a stronger bindingaffinity for an antigen from a first subject species than it has for ahomologue of that antigen from a second subject species. Normally, theantibody “bind specifically” to a human antigen (i.e., has a bindingaffinity (Kd) value of no more than about 1×10⁻⁷ M, preferably no morethan about 1×10⁻⁸ M and most preferably no more than about 1×10⁻⁹ M) buthas a binding affinity for a homologue of the antigen from a secondsubject species which is at least about 50 fold, or at least about 500fold, or at least about 1000 fold, weaker than its binding affinity forthe human antigen. The antibody can be of any of the various types ofantibodies as defined above, but preferably is a humanized or humanantibody (Queen et al., U.S. Pat. Nos. 5,530,101, 5,585,089; 5,693,762;and 6,180,370).

The present invention provides an “antibody variant,” which refers to anamino acid sequence variant of an antibody wherein one or more of theamino acid residues have been modified. Such variant necessarily haveless than 100% sequence identity or similarity with the amino acidsequence having at least 75%, amino acid sequence identity or similaritywith the amino acid sequence of either the heavy or light chain variabledomain of the antibody, more preferably at least 80%, more preferably atleast 85%, more preferably at least 90%, and most preferably at least95%. Since the method of the invention applies equally to bothpolypeptides, antibodies and fragments thereof, these terms aresometimes employed interchangeably.

The term “variable” in the context of variable domain of antibodiesrefers to the fact that certain portions of the variable domains differextensively in sequence among antibodies and are used in the binding andspecificity of each particular antibody for its particular antigen.However, the variability is not evenly distributed through the variabledomains of antibodies. It is concentrated in three segments calledcomplementarity determining regions (CDRs) also known as hypervariableregions both in the light chain and the heavy chain variable domains.There are at least two techniques for determining CDRs: (1) an approachbased on cross-species sequence variability (i.e., Kabat et al.,Sequences of Proteins of Immunological Interest (National Institute ofHealth, Bethesda, Md. 1987); and (2) an approach based oncrystallographic studies of antigen-antibody complexes (Chothia, C. etal. (1989), Nature 342: 877). The more highly conserved portions ofvariable domains are called the framework (FR). The variable domains ofnative heavy and light chains each comprise four FR regions, largelyadopting a β-Sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen-binding site of antibodies (see Kabat etal.) The constant domains are not involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and Fv fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding fragments which are capable of crosslinkingantigen, and a residual other fragment (which is termed pFc′).Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “functional fragment” withrespect to antibodies, refers to Fv, F(ab) and F(ab′)₂ fragments.

An “Fv” fragment is the minimum antibody fragment that contains acomplete antigen recognition and binding site. This region consists of adimer of one heavy and one light chain variable domain in a tight,non-covalent association (V_(H)-V_(L) dimer). It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six CDRs confer antigen-binding specificity to theantibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen, although at a lower affinity than the entirebinding site.

The Fab fragment [also designated as F(ab)] also contains the constantdomain of the light chain and the first constant domain (CH1) of theheavy chain. Fab′ fragments differ from Fab fragments by the addition ofa few residues at the carboxyl terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region. Fab′-SHis the designation herein for Fab′ in which the cysteine residue(s) ofthe constant domains have a free thiol group. F(ab′) fragments areproduced by cleavage of the disulfide bond at the hinge cysteines of theF(ab′)₂ pepsin digestion product. Additional chemical couplings ofantibody fragments are known to those of ordinary skill in the art.

The present invention further provides monoclonal antibodies, polyclonalantibodies as well as humanized antibody. In general, to generateantibodies, an isolated peptide is used as an immunogen and isadministered to a mammalian organism, such as a rat, rabbit or mouse.The full-length protein, an antigenic peptide fragment or a fusionprotein of the PCAT protein can be used. Particularly importantfragments are those covering functional domains, some but not all theexamples of the domains are identified in Tables. Many methods are knownfor generating and/or identifying antibodies to a given target peptide.Several such methods are described by Harlow, Antibodies, Cold SpringHarbor Press, (1989).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In additional to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”antibody indicates the character of the antibody as being obtained froma substantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler and Milstein, Nature 256, 495 (1975), or may be madeby recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567.The monoclonal antibodies for use with the present invention may also beisolated from phage antibody libraries using the techniques described inClackson et al., Nature 352: 624-628 (1991), as well as in Marks et al.,J. Mol. Biol. 222: 581-597 (1991). For detailed procedures for making amonoclonal antibody, see the Example below.

“Humanized” forms of non-human (e.g. murine or rabbit) antibodies arechimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. For the most part, humanized antibodies arehuman immunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibody may comprise residues, which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., Nature 321,522-525 (1986); Reichmann et al., Nature 332, 323-327 (1988) and Presta,Curr. Op. Struct. Biol. 2, 593-596 (1992).

Polyclonal antibodies may be prepared by any known method ormodifications of these methods including obtaining antibodies frompatients. For example, a complex of an immunogen such as PCAT proteins,peptides or fragments thereof and a carrier protein is prepared and ananimal is immunized by the complex according to the same manner as thatdescribed with respect to the above monoclonal antibody preparation andthe description in the Example. A serum or plasma containing theantibody against the protein is recovered from the immunized animal andthe antibody is separated and purified. The gamma globulin fraction orthe IgG antibodies can be obtained, for example, by use of saturatedammonium sulfate or DEAE Sephadex, or other techniques known to thoseskilled in the art.

The antibody titer in the antiserum can be measured according to thesame manner as that described above with respect to the supernatant ofthe hybridoma culture. Separation and purification of the antibody canbe carried out according to the same separation and purification methodof antibody as that described with respect to the above monoclonalantibody and in the Example.

The protein used herein as the immunogen is not limited to anyparticular type of immunogen. In one aspect, antibodies are preferablyprepared from regions or discrete fragments of the PCAT proteins.Antibodies can be prepared from any region of the peptide as describedherein. In particular, they are selected from a group consisting of SEQID NOS: 703-873 and fragments of SEQ ID NOS: 1-288. An antigenicfragment will typically comprise at least 8 contiguous amino acidresidues. The antigenic peptide can comprise, however, at least 10, 12,14, 16 or more amino acid residues. Such fragments can be selected on aphysical property, such as fragments correspond to regions that arelocated on the surface of the protein, e.g., hydrophilic regions or canbe selected based on sequence uniqueness.

Antibodies may also be produced by inducing production in the lymphocytepopulation or by screening antibody libraries or panels of highlyspecific binding reagents as disclosed in Orlandi et al. (1989; ProcNatl Acad Sci 86: 3833-3837) or Winter et al. (1991; Nature 349:293-299). A protein may be used in screening assays of phagemid orB-lymphocyte immunoglobulin libraries to identify antibodies having adesired specificity. Numerous protocols for competitive binding orimmunoassays using either polyclonal or monoclonal antibodies withestablished specificities are well known in the art. Smith G. P., 1991,Curr. Opin. Biotechnol. 2: 668-673.

The antibodies of the present invention can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In a particular, such phage can be utilized to display antigen-bindingdomains expressed from a repertoire or combinatorial antibody library(e.g., human or murine). Phage expressing an antigen binding domain thatbinds the antigen of interest can be selected or identified withantigen, e.g., using labeled antigen or antigen bound or captured to asolid surface or bead. Phage used in these methods are typicallyfilamentous phage including fd and M13 binding domains expressed fromphage with Fab, Fv or disulfide stabilized Fv antibody domainsrecombinantly fused to either the phage gene III or gene VIII protein.Examples of phage display methods that can be used to make theantibodies of the present invention include those disclosed in Brinkmanet al., J. Immunol Methods 182: 41-50 (1995); Ames et al., J. Immunol.Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al.,Advances in Immunology 57: 191-280 (1994); PCT application No.PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047;WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727;5,733,743 and 5,969,108; each of which is incorporated herein byreference in its entirety.

Antibody can be also made recombinantly. When using recombinanttechniques, the antibody variant can be produced intracellularly, in theperiplasmic space, or directly secreted into the medium. If the antibodyvariant is produced intracellularly, as a first step, the particulatedebris, either host cells or lysed fragments, is removed, for example,by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163-167 (1992) describe a procedure for isolating antibodies which aresecreted to the periplasmic space of E. coli. Briefly, cell paste isthawed in the presence of sodium acetate (pH 3.5), EDTA, andphenylmethylsulfonylfluoride (PMSF) over about 30 minutes. Cell debriscan be removed by centrifugation. Where the antibody variant is secretedinto the medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibodies or antigen binding fragments may also be produced bygenetic engineering. The technology for expression of both heavy andlight cain genes in E. coli is the subject the following PCT patentapplications; publication number WO 901443, WO901443, and WO 9014424 andin Huse et al., 1989 Science 246: 1275-1281. The general recombinantmethods are well known in the art.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human delta. 1, .delta.2or .delta.4 heavy chains (Lindmark et al., J. Immunol Meth. 62: 1-13(1983)). Protein G is recommended for all mouse isotypes and for human.delta.3 (Guss et al., EMBO J. 5: 1567-1575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a CH3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE™ chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in Table 1 indicate that thePCATs of the present invention are expressed at differential level invarious pancreatic cell lines, for example SEQ ID NOS: 1-119 areoverexpressed in all tested cell lines, whereas other proteins orpeptides are underexpressed in selected cell lines. In one example,protein (SEQ ID NO: 120) or peptide (SEQ ID NO: 761) is overexpressed inone cell line (HPAC), yet underexpressed in another cell line(Su.86.86). Further, such antibodies can be used to detect protein insitu, in vitro, or in a cell lysate or supernatant in order to evaluatethe abundance and pattern of expression. Also, such antibodies can beused to assess abnormal tissue distribution or abnormal expressionduring development or progression of a biological condition. Antibodydetection of circulating fragments of the full length protein can beused to identify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in Table 1 indicatesexpression in human pancreatic cell lines. If a disorder ischaracterized by a specific mutation in the protein, antibodies specificfor this mutant protein can be used to assay for the presence of thespecific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in Table 1 indicates expression in humanpancreatic cell lines. The diagnostic uses can be applied, not only ingenetic testing, but also in monitoring a treatment modality.Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy. More detection and diagnostic methods aredescribed in detail below.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools, as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in Table 1 indicates expression in human pancreatic cell lines.Thus, where a specific protein has been correlated with expression in aspecific tissue, antibodies that are specific for this protein can beused to identify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the PCAT peptide to a binding partnersuch as a substrate or another antibody binding to the PCAT. These usescan also be applied in a therapeutic context in which treatment involvesinhibiting the protein's function. An antibody can be used, for example,to block binding, thus modulating (agonizing or antagonizing) thepeptides activity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated with a cell or cell membrane. More therapeutics methods aredescribed in detail below.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornucleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a PCAT peptide or protein of the present invention. Suchnucleic acid molecules will consist of, consist essentially of, orcomprise a nucleotide sequence that encodes one of the PCAT peptides ofthe present invention, an allelic variant thereof, or an ortholog orparalog thereof. The nucleic acid molecules and the fragments thereof ofthe present invention pertains, however, are not to be construed asencompassing fragments that may be disclosed publicly prior to thepresent invention.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5 KB, 4KB, 3 KB, 2 KB, or 1 KB or less, particularly contiguous peptideencoding sequences and peptide encoding sequences within the same genebut separated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

The present invention further provides nucleic acid molecules thatcomprise the nucleotide sequences shown in Table 2, (SEQ ID NOS:289-702), or any nucleic acid molecule that encodes the protein providedin Table 2, (SEQ ID NOS: 1-288). A nucleic acid molecule comprises anucleotide sequence when the nucleotide sequence is at least part of thefinal nucleotide sequence of the nucleic acid molecule. In such afashion, the nucleic acid molecule can be only the nucleotide sequenceor have additional nucleic acid residues, such as nucleic acid residuesthat are naturally associated with it or heterologous nucleotidesequences. Such a nucleic acid molecule can have a few additionalnucleotides or can comprise several hundred or more additionalnucleotides. A brief description of how various types of these nucleicacid molecules can be readily made/isolated is provided below.

In Table 2, human transcript sequences are provided. As discussed below,some of the non-coding regions, particularly gene regulatory elementssuch as promoters, are useful for a variety of purposes, e.g. control ofheterologous gene expression, target for identifying gene activitymodulating compounds, and are particularly claimed as fragments of thegenomic sequence provided herein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the PCAT peptide alone, thesequence encoding the mature peptide and additional coding sequences,such as a leader or secretory sequence (e.g., a pre-pro or pro-proteinsequence), the sequence encoding the mature peptide, with or without theadditional coding sequences, plus additional non-coding sequences, forexample introns and non-coding 5′ and 3′ sequences such as transcribedbut non-translated sequences that play a role in transcription, mRNAprocessing (including splicing and polyadenylation signals), ribosomebinding and stability of mRNA. In addition, the nucleic acid moleculemay be fused to a marker sequence encoding, for example, a peptide thatfacilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the PCAT proteins of thepresent invention that are described above. Such nucleic acid moleculesmay be naturally occurring, such as allelic variants (same locus),paralogs (different locus), and orthologs (different organism), or maybe constructed by recombinant DNA methods or by chemical synthesis. Suchnon-naturally occurring variants may be made by mutagenesis techniques,including those applied to nucleic acid molecules, cells, or organisms.Accordingly, as discussed above, the variants can contain nucleotidesubstitutions, deletions, inversions and insertions. Variation can occurin either or both the coding and non-coding regions. The variations canproduce both conservative and non-conservative amino acid substitutions.

The present invention further provides non-coding fragments of thenucleic acid molecules provided in Tables 1 and 2. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thegenomic sequence.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of thegene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Table2 or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions is hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45 C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65 C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in Tables 1 and 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in Tables1 and 2.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Tables. Accordingly, it could bederived from. 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided in Table 1 indicate that thePCATs of the present invention are expressed at differential level invarious pancreatic cell lines, for example SEQ ID NOS: 1-119 areoverexpressed in all tested cell lines, whereas other proteins orpeptides are underexpressed in selected cell lines. In one example,protein (SEQ ID NO: 120) or peptide (SEQ ID NO: 761) is overexpressed inone cell line (HPAC), yet underexpressed in another cell line(Su.86.86). Accordingly, the probes can be used to detect the presenceof, or to determine levels of, a specific nucleic acid molecule incells, tissues, and in organisms. The nucleic acid whose level isdetermined can be DNA or RNA. Accordingly, probes corresponding to thepeptides described herein can be used to assess expression and/or genecopy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease inPCAT protein expression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a PCAT protein, such as by measuring alevel of a PCAT-encoding nucleic acid in a sample of cells from asubject e.g., mRNA or genomic DNA, or determining if a PCAT gene hasbeen mutated. Experimental data as provided in Table 1 indicate that thePCATs of the present invention are expressed at differential level invarious pancreatic cell lines, for example SEQ ID NOS: 1-119 areoverexpressed in all tested cell lines, whereas other proteins orpeptides are underexpressed in selected cell lines. In one example,protein (SEQ ID NO: 120) or peptide (SEQ ID NO: 761) is overexpressed inone cell line (HPAC), yet underexpressed in another cell line(Su.86.86). More detection and diagnostic methods are described indetail below.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate PCAT nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe PCAT gene, particularly biological and pathological processes thatare mediated by the PCAT in cells and tissues that express it.Experimental data as provided in Table 1 indicates expression in humanpancreatic cell lines. The method typically includes assaying theability of the compound to modulate the expression of the PCAT nucleicacid and thus identifying a compound that can be used to treat adisorder characterized by undesired PCAT nucleic acid expression. Theassays can be performed in cell-based and cell-free systems. Cell-basedassays include cells naturally expressing the PCAT nucleic acid orrecombinant cells genetically engineered to express specific nucleicacid sequences.

The assay for PCAT nucleic acid expression can involve direct assay ofnucleic acid levels, such as mRNA levels, or on collateral compoundsinvolved in the signal pathway. Further, the expression of genes thatare up- or down-regulated in response to the PCAT protein signal pathwaycan also be assayed. In this embodiment the regulatory regions of thesegenes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of PCAT gene expression can be identified in a methodwherein a cell is contacted with a candidate compound or agent and theexpression of mRNA determined. The level of expression of PCAT mRNA inthe presence of the candidate compound or agent is compared to the levelof expression of PCAT mRNA in the absence of the candidate compound oragent. The candidate compound can then be identified as a modulator ofnucleic acid expression based on this comparison and be used, forexample to treat a disorder characterized by aberrant nucleic acidexpression. When expression of mRNA is statistically significantlygreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of nucleic acidexpression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound or an agent identified through drugscreening as a gene modulator to modulate PCAT nucleic acid expressionin cells and tissues that express the PCAT. Experimental data asprovided in Table 1 indicate that the PCATs of the present invention areexpressed at differential level in various pancreatic cell lines, forexample SEQ ID NOS: 1-119 are overexpressed in all tested cell lines,whereas other proteins or peptides are underexpressed in selected celllines. In one example, protein (SEQ ID NO: 120) or peptide (SEQ ID NO:761) is overexpressed in one cell line (HPAC), yet underexpressed inanother cell line (Su.86.86). Modulation includes both up-regulation(i.e. activation or agonization) or down-regulation (suppression orantagonization) or nucleic acid expression.

Alternatively, a modulator for nucleic acid expression can be a smallmolecule or drug identified using the screening assays described hereinas long as the drug or small molecule inhibits the PCAT nucleic acidexpression in the cells and tissues that express the protein.Experimental data as provided in Table 1 indicates expression in humanpancreatic cell lines.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds or agents on the expression oractivity of the PCAT gene in clinical trials or in a treatment regimen.Thus, the gene expression pattern can serve as a barometer for thecontinuing effectiveness of treatment with the compound, particularlywith compounds to which a patient can develop resistance. The geneexpression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in PCAT nucleic acid expression, and particularly inqualitative changes that lead to pathology. The nucleic acid moleculescan be used to detect mutations in PCAT genes and gene expressionproducts such as mRNA. The nucleic acid molecules can be used ashybridization probes to detect naturally occurring genetic mutations inthe PCAT gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification Detectionof a mutated form of the PCAT gene associated with a dysfunctionprovides a diagnostic tool for an active disease or susceptibility todisease when the disease results from overexpression, underexpression,or altered expression of a PCAT protein.

Individuals carrying mutations in the PCAT gene can be detected at thenucleic acid level by a variety of techniques. Genomic DNA can beanalyzed directly or can be amplified by using PCR prior to analysis.RNA or cDNA can be used in the same way. In some uses, detection of themutation involves the use of a probe/primer in a polymerase chainreaction (PCR) (see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), suchas anchor PCR or RACE PCR, or, alternatively, in a ligation chainreaction (LCR) (see, e.g., Landegran et al., Science 241: 1077-1080(1988); and Nakazawa et al., PNAS 91: 360-364 (1994)), the latter ofwhich can be particularly useful for detecting point mutations in thegene (see Abravaya et al., Nucleic Acids Res. 23: 675-682 (1995)). Thismethod can include the steps of collecting a sample of cells from apatient, isolating nucleic acid (e.g., genomic, mRNA or both) from thecells of the sample, contacting the nucleic acid sample with one or moreprimers which specifically hybridize to a gene under conditions suchthat hybridization and amplification of the gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. Deletions and insertions can be detected by achange in size of the amplified product compared to the normal genotype.Point mutations can be identified by hybridizing amplified DNA to normalRNA or antisense DNA sequences.

Alternatively, mutations in a PCAT gene can be directly identified, forexample, by alterations in restriction enzyme digestion patternsdetermined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutant PCATgene and a wild-type gene can be determined by direct DNA sequencing. Avariety of automated sequencing procedures can be utilized whenperforming the diagnostic assays (Naeve, C. W., (1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCTInternational Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36: 127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38: 147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230: 1242 (1985));Cotton et al., PNAS 85: 4397 (1988); Saleeba et al., Meth. Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86: 2766 (1989); Cotton etal., Mutat. Res. 285: 125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9: 73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313: 495 (1985)). Examples of other techniques for detectingpoint mutations include selective oligonucleotide hybridization,selective amplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the PCAT gene in anindividual in order to select an appropriate compound or dosage regimenfor treatment.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol PCAT gene expression in cells, tissues, and organisms. A DNAantisense nucleic acid molecule is designed to be complementary to aregion of the gene involved in transcription, preventing transcriptionand hence production of PCAT protein. An antisense RNA or DNA nucleicacid molecule would hybridize to the mRNA and thus block translation ofmRNA into PCAT protein.

The nucleic acid of the present invention may also be used tospecifically suppress gene expression by methods such as RNAinterference (RNAi), which may also include cosuppression and quelling.This and antisense RNA or DNA of gene suppression are well known in theart. A review of this technique is found in Science 288: 1370-1372,2000. RNAi also operates on a post-transcriptional level and is sequencespecific, but suppresses gene expression far more efficiently thanantisense RNA. RNAi fragments, particularly double-stranded (ds) RNAi,can be also used to generate loss-of-function phenotypes.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of PCAT nucleic acid. Accordingly,these molecules can treat a disorder characterized by abnormal orundesired PCAT nucleic acid expression. This technique involves cleavageby means of ribozymes containing nucleotide sequences complementary toone or more regions in the mRNA that attenuate the ability of the mRNAto be translated. Possible regions include coding regions andparticularly coding regions corresponding to the catalytic and otherfunctional activities of the PCAT protein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in PCAT gene expression.Thus, recombinant cells, which include the patient's cells that havebeen engineered ex vivo and returned to the patient, are introduced intoan individual where the cells produce the desired PCAT protein to treatthe individual.

The invention also encompasses kits for detecting the presence of a PCATnucleic acid in a biological sample. Experimental data as provided inTable 1 indicate that the PCATs of the present invention are expressedat differential level in various pancreatic cell lines, for example SEQID NOS: 1-119 are overexpressed in all tested cell lines, whereas otherproteins or peptides are underexpressed in selected cell lines. In oneexample, protein (SEQ ID NO: 120) or peptide (SEQ ID NO: 761) isoverexpressed in one cell line (HPAC), yet underexpressed in anothercell line (Su.86.86). For example, the kit can comprise reagents such asa labeled or labelable nucleic acid or agent capable of detecting PCATnucleic acid in a biological sample; means for determining the amount ofPCAT nucleic acid in the sample; and means for comparing the amount ofPCAT nucleic acid in the sample with a standard. The compound or agentcan be packaged in a suitable container. The kit can further compriseinstructions for using the kit to detect PCAT protein mRNA or DNA.

Vectors/Host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequences to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terminalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(2001).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 3rd. ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (2001).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodologies. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein; increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enteroenzyme. Typical fusion expression vectors includepGEX (Smith et al., Gene 67: 31-40 (1988)), pMAL (New England Biolabs,Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuseglutathione S-transferase (GST), maltose E binding protein, or proteinA, respectively, to the target recombinant protein. Examples of suitableinducible non-fusion E. coli expression vectors include pTrc (Amann etal., Gene 69: 301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185: 60-89 (1990)).Recombinant protein expression can be maximized in host bacteria byproviding a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20: 2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234 (1987)), pMFa (Kurjan et al., Cell 30: 933-943 (1982)), pJRY88(Schultz et al., Gene 54: 113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology 170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329: 840 (1987)) and pMT2PC (Kaufman et al., EMBOJ. 6: 187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.3rd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2001.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 3rd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction Viral vectors can be replication-competent orreplication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell-free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which may be difficult toachieve with multi-transmembrane domain containing proteins such asPCATs, appropriate secretion signals are incorporated into the vector.The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

Where the peptide is not secreted into the medium, the protein can beisolated from the host cell by standard disruption procedures, includingfreeze thaw, sonication, mechanical disruption, use of lysing agents andthe like. The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing a PCATprotein or peptide that can be further purified to produce desiredamounts of PCAT protein or fragments. Thus, host cells containingexpression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe PCAT protein or PCAT protein fragments, such as those describedabove as well as other formats known in the art. Thus, a recombinanthost cell expressing a native PCAT protein is useful for assayingcompounds that stimulate or inhibit PCAT protein function.

Host cells are also useful for identifying PCAT protein mutants in whichthese functions are affected. If the mutants naturally occur and giverise to a pathology, host cells containing the mutations are useful toassay compounds that have a desired effect on the mutant PCAT protein(for example, stimulating or inhibiting function) which may not beindicated by their effect on the native PCAT protein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a PCAT protein andidentifying and evaluating modulators of PCAT protein activity. Otherexamples of transgenic animals include non-human primates, sheep, dogs,cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the PCAT protein nucleotidesequences can be introduced as a transgene into the genome of anon-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the PCAT protein to particular cells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89: 6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251: 1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385: 810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter Go phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyst and then transferred to pseudopregnant female foster animal.The offspring born of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, PCAT protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo PCATprotein function, including substrate interaction, the effect ofspecific mutant PCAT proteins on PCAT protein function and substrateinteraction, and the effect of chimeric PCAT proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more PCAT proteinfunctions.

Detection and Diagnosis

The present invention provides a method for detecting PCAT nucleicacids, proteins, peptides and fragments thereof that are differentiallyexpressed in pancreatic diseases in a test sample, preferably in abiological sample.

The present invention further provides a method for diagnosing thepancreatic diseases, by detecting the nucleic acids, proteins, peptidesand fragments thereof. The further embodiment includes but is notlimited to, monitoring the disease prognosis (recurrance), diagnosingdisease stage, preventing the disease and treating the disease.

As used herein, a “biological sample” can be collected from tissues,blood, sera, cell lines or biological fluids such as, plasma,interstitial fluid, urine, cerebrospinal fluid, and the like, containingcells. In preferred embodiments, a biological sample comprises cells ortissues suspected of having diseases (e.g., cells obtained from abiopsy).

As used herein, a “differential level” is defined as the level of PCATprotein or nucleic acids in a test sample either above or below thelevel of the ones in control samples, wherein the level of controlsamples is obtained from a control cell line, a normal tissue or bodyfluids, or combination thereof, or from a healthy subject. While theprotein is overexpressed, the expression of PCAT is preferably greaterthan about 20%, or preferably greater than about 30%, and mostpreferably greater than about 50% or more of pancreatic disease sample,at a level that is at least two fold, and preferably at least five fold,greater than the level of expression in control samples, as determinedusing a representative assay provided herein. While the protein isunderexpressed, the expression of PCAT is preferably less than about20%, or preferably less than 30%, and most preferably less than about50% or more of the pancreatic disease sample, at a level that is atleast 0.5 fold, and preferably at least 0.2 fold less than the level ofthe expression in control samples, as determined using a representativeassay provided herein.

As used herein, a “subject” can be a mammalian subject or non mammaliansubject, preferably, a mammalian subject. A mammalian subject can behuman or non-human, preferably human. A healthy subject is defined as asubject without detectable pancreatic diseases or pancreatic associateddiseases by using conventional diagnostic methods.

As used herein, the “diseases” include pancreatic diseases andpancreatic associated diseases. Preferably, the pancreatic disease ispancreatic cancer.

As used herein, “cancer” includes epithelial-cell related cancers, forexample pancreatic, lung, colon, prostate, ovarian, breast, bladderrenal, hepatocellular, pharyngeal and gastric cancers.

Nucleic Acid Detections

The present invention is not limited to the detection methods describedabove. Any suitable detection method that allows for the specificdetection of pancreatic disease cells, tissues or organs may beutilized. For example, in some embodiments, the expression of RNAcorresponding to a PCAT gene is detected by hybridization to anantisense oligonucleotide (described below). In other embodiments, RNAexpression is detected by hybridization assays such as Northern blots,RNase assays, reverse transcriptase PCR amplification, and the like. Onepreferred detection method is using RT PCR by using TaqMan technology(ABI, Foster City, Calif.).

In another embodiment, the present invention provides a method fordiagnosing or detecting pancreatic diseases in a subject comprising:determining the level of one or more PCAT nucleic acid molecules or anyfragment(s) thereof in a test sample from said subject, wherein saidPCAT nucleic acid molecule(s) comprises a sequence selected from a groupconsisting of SEQ ID NOS: 289-702 and a combination thereof; wherein adifferential level of said PCAT nucleic acid molecule(s) relative to thelevel of said nucleic acid molecule(s) in a test sample from a healthysubject, or the level established for a healthy subject, is indicativeof pancreatic diseases.

In another embodiment, the detecting or diagnosing method comprisesdetermining level of differential expression of 2, 4, 8, 10, 20 or morenucleic acid molecules, preferably, the nucleic acid molecules compriseor consists of a sequence selected from the group consisting of SEQ IDNOS: 289-702 and combination thereof.

In further embodiments of the present invention, the presence ofparticular sequences in the genome of a subject is detected. Suchsequences include PCAT sequences associated with abnormal expression ofPCAT (e.g., overexpression or expression at a physiologicalinappropriate time). These sequences include polymorphisms, includingpolymorphisms in the transcribed sequence (e.g., that effect PCATprocessing and/or translation) and regulatory sequences such aspromoters, enhances, repressors, and the like. These sequences may alsoinclude polymorphisms in genes or control sequences associated withfactors that affect expression such as transcription factors, and thelike. Any suitable method for detecting and/or identifying thesesequences is within the scope of the present invention including, butnot limited to, nucleic acid sequencing, hybridization assays (e.g.,Southern blotting), single nucleotide polymorphism assays (See e.g.,U.S. Pat. No. 5,994,069, herein incorporated by reference in itsentirety), and the like.

Protein Detections

The present invention provides methods for diagnosing or detecting thedifferential presence of PCAT proteins. In some embodiments (e.g., wherePCATs are overexpressed in diseased cells), PCAT proteins are detecteddirectly. In other embodiments (e.g., where the presence of a PCATs areunderexpressed), PCATs to the disease antigens are detectednon-existence.

The diagnostic methods of the present invention find utility in thediagnosis and characterization of diseases. For example, the presence ofan antibody to a specific protein may be indicative of a cancer ordisease. In addition, certain PCAT may be indicative of a specific stageor sub-type of the same cancer or disease.

The information obtained is also used to determine prognosis andappropriate course of treatment. For example, it is contemplated thatindividuals with a specific PCAT expression or stage of pancreaticdiseases may respond differently to a given treatment that individualslacking the PCAT expression. The information obtained from thediagnostic methods of the present invention thus provides for thepersonalization of diagnosis and treatment.

In one embodiment, the present invention provides a method formonitoring pancreatic diseases treatment in a subject comprising:determining the level of one or more PCAT proteins or any fragment(s) orpeptide(s) thereof in a test sample from said subject, wherein said PCATprotein(s) comprises a sequence selected from a group consisting of SEQID NOS: 1-288, SEQ ID NOS: 703-873 and a combination thereof; wherein anlevel of said PCAT protein(s) similar to the level of said protein(s) ina test sample from a healthy subject, or the level established for ahealthy subject, is indicative of successful treatment.

In another embodiment, the present invention provides a method fordiagnosing recurrence of pancreatic diseases following successfultreatment in a subject comprising: determining the level of one or morePCAT proteins or any fragment(s) or peptide(s) thereof in a test samplefrom said subject, wherein said PCAT protein(s) comprises a sequenceselected from a group consisting of SEQ ID NOS: 1-288, SEQ ID NOS:703-873 and a combination thereof; wherein a changed level of said PCATprotein(s) relative to the level of said protein(s) in a test samplefrom a healthy subject, or the level established for a healthy subject,is indicative of recurrence of pancreatic diseases.

In yet another embodiment, the present invention provides a method fordiagnosing or detecting pancreatic diseases in a subject comprising:determining the level of one or more PCAT proteins or any fragment(s) orpeptides thereof in a test sample from said subject, wherein said PCATprotein(s) comprises a sequence selected from a group consisting of SEQID NOS: 1-288, SEQ ID NOS: 703-873 and a combination thereof; wherein adifferential level of said PCAT protein(s) relative to the level of saidprotein(s) in a test sample from a healthy subject, or the levelestablished for a healthy subject, is indicative of pancreatic diseases.

The detecting or diagnosing method comprises determining level ofdifferential expression of 2, 4, 8, 10, 20 or more proteins, preferably,the proteins are selected from a group consisting of SEQ ID NOS: 1-288and combination thereof.

Further, the detecting or diagnosing method comprises determining levelof differential expression of 5, 10, 15, 20, 40, 60, 80, 100 or morePCAT peptides, preferably the peptides are selected from the groupconsisting of SEQ ID NOS: 703-873 and combination thereof.

These methods are also useful for diagnosing diseases that showdifferential protein expression. As describe earlier, normal, control orstandard values or level established from a healthy subject for proteinexpression are established by combining body fluids or tissue, cellextracts taken from a normal healthy mammalian or human subject withspecific antibodies to a protein under conditions for complex formation.Standard values for complex formation in normal and diseased tissues areestablished by various methods, often photometric means. Then complexformation as it is expressed in a subject sample is compared with thestandard values. Deviation from the normal standard and toward thediseased standard provides parameters for disease diagnosis or prognosiswhile deviation away from the diseased and toward the normal standardmay be used to evaluate treatment efficacy.

In yet another embodiment, the present invention provides a detection ordiagonistic method of PCATs by using LC/MS. The proteins from cells areprepared by methods known in the art (for example, R. Aebersold NatureBiotechnology Volume 21 Number 6 Jun. 2003). The differential expressionof proteins in disease and healthy samples are quantitated using MassSpectrometry and ICAT (Isotope Coded Affinity Tag) labeling, which isknown in the art. ICAT is an isotope label technique that allows fordiscrimination between two populations of proteins, such as a healthyand a disease sample. The LC/MS spectra are collected for the labeledsamples. The raw scans from the LC/MS instrument are subjected to peakdetection and noise reduction software. Filtered peak lists are thenused to detect ‘features’ corresponding to specific peptides from theoriginal sample(s). Features are characterized by their mass/charge,charge, retention time, isotope pattern and intensity.

The intensity of a peptide present in both healthy and disease samplescan be used to calculate the differential expression, or relativeabundance, of the peptide. The intensity of a peptide found exclusivelyin one sample can be used to calculate a theoretical expression ratiofor that peptide (singleton). Expression ratios are calculated for eachpeptide of each replicate of the experiment (Table 1). Thusoverexpression or under expression of PCAT protein or peptide aresimilar to the expression pattern in Table 1 in a test subject indicatesthe likelihood of having pancreatic diseases or diseases associated withpancreas.

Immunological methods for detecting and measuring complex formation as ameasure of protein expression using either specific polyclonal ormonoclonal antibodies are known in the art. Examples of such techniquesinclude enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays(RIAs), fluorescence-activated cell sorting (FACS) and antibody arrays.Such immunoassays typically involve the measurement of complex formationbetween the protein and its specific antibody. These assays and theirquantitation against purified, labeled standards are well known in theart (Ausubel, supra, unit 10.1-10.6). A two-site, monoclonal-basedimmunoassay utilizing antibodies reactive to two non-interferingepitopes is preferred, but a competitive binding assay may be employed(Pound (1998) Immunochemical Protocols, Humana Press, Totowa N.J.). Moreimmunological detections are described in the sections below.

Antibody Detections

Antibodies are useful to detect the presence of one of the proteins orfragments thereof, peptides of the present invention in cells or tissuesto determine the pattern of expression of the proteins among varioustissues in an organism and over the course of normal development.

Further, as described above, the antibodies can be used to assessexpression in disease states such as in active stages of the disease orin an individual with a predisposition toward disease related to theprotein's function. The antibodies can also be used to assess normal andaberrant subcellular localization of cells in the various tissues in anorganism.

Detection on a protein by an antibody can be facilitated by coupling(i.e., physically linking) the antibody to a detectable substance.Examples of detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials, bioluminescentmaterials, and radioactive materials (see below). The antibodies mayalso be useful in diagnostic assays, e.g., for detecting expression ofan antigen, for example PCAT protein, peptide or fragment thereof, inspecific cells, tissues, blood, serum or body fluids.

For diagnostic applications, the antibody or its variant typically willbe labeled with a detectable moiety. Numerous labels are available whichcan be generally grouped into the following categories:

(a) Radioisotopes, such as ³⁶ S. ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibodyvariant can be labeled with the radioisotope using the techniquesdescribed in Current Protocols in Immunology, vol 1-2, Coligen et al.,Ed., Wiley-Interscience, New York, Pubs. (1991) for example andradioactivity can be measured using scintillation counting.

(b) Fluorescent labels such as rare earth chelates (europium chelates)or fluorescein and its derivatives, rhodamine and its derivatives,dansyl, Lissamine, phycoerythrin and Texas Red are available. Thefluorescent labels can be conjugated to the antibody variant using thetechniques disclosed in Current Protocols in Immunology, supra, forexample. Fluorescence can be quantified using a fluorimeter.

(c) Various enzyme-substrate labels are available and U.S. Pat. Nos.4,275,149, and 4,318,980 provides a review of some of these. The enzymegenerally catalyzes a chemical alteration of the chromogenic substratewhich can be measured using various techniques. For example, the enzymemay catalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme may alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and may then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for Use inEnzyme Immunoassay, in Methods in Enzyme. (Ed. J. Langone & H. VanVunakis), Academic press, New York, 73: 147-166 (1981).

Sometimes, the label is indirectly conjugated with the antibody. Theskilled artisan will be aware of various techniques for achieving this.For example, the antibody can be conjugated with biotin and any of thethree broad categories of labels mentioned above can be conjugated withavidin, or vice versa. Biotin binds selectively to avidin and thus, thelabel can be conjugated with the antibody in this indirect manner.Alternatively, to achieve indirect conjugation of the label with theantibody, the antibody is conjugated with a small hapten (e.g. digloxin)and one of the different types of labels mentioned above is conjugatedwith an anti-hapten antibody (e.g. anti-digloxin antibody). Thus,indirect conjugation of the label with the antibody can be achieved.

In another embodiment of the invention, the antibody needs not belabeled, and the presence thereof can be detected using a labeledantibody, which binds to the antibody.

The antibodies of the present invention may be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc. 1987).

The biological samples can then be tested directly for the presence ofPCAT by assays (e.g., ELISA or radioimmunoassay) and format (e.g.,microwells, dipstick as described in International Patent Publication WO93/03367). Alternatively, proteins in the sample can be size separated(e.g., by polyacrylamide gel electrophoresis (PAGE)), in the presence orabsence of sodium dodecyl sulfate (SDS), and the presence of PCATdetected by immunoblotting (e.g., Western blotting). Immunoblottingtechniques are generally more effective with antibodies generatedagainst a peptide corresponding to an epitope of a protein, and hence,are particularly suited to the present invention.

Antibody binding is detected by techniques known in the art (e.g.,radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (e.g., usingcolloidal gold, enzyme or radioisotope labels, for example), Westernblots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays, etc.), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

In one embodiment, antibody binding is detected by detecting a label onthe primary antibody. In another embodiment, the primary antibody isdetected by detecting binding of a secondary antibody or reagent to theprimary antibody. In a further embodiment, the secondary antibody islabeled. Many means are known in the art for detecting binding in animmunoassay and are within the scope of the present invention. As iswell known in the art, the immunogenic peptide should be provided freeof the carrier molecule used in any immunization protocol. For example,if the peptide was conjugated to KLH, it may be conjugated to BSA, orused directly, in a screening assay. In some embodiments, an automateddetection assay is utilized. Methods for the automation of immunoassaysare well known in the art (See e.g., U.S. Pat. Nos. 5,885,530,4,981,785, 6,159,750, and 5,358,691; each of which is hereinincorporated by reference). In some embodiments, the analysis andpresentation of results is also automated. For example, in someembodiments, software that generates a prognosis based on the presenceor absence of a series of antigens is utilized.

Competitive binding assays rely on the ability of a labeled standard tocompete with the test sample for binding with a limited amount ofantibody. The amount of antigen in the test sample is inverselyproportional to the amount of standard that becomes bound to theantibodies. To facilitate determining the amount of standard thatbecomes bound, the antibodies generally are insolubilized before orafter the competition. As a result, the standard and test sample thatare bound to the antibodies may conveniently be separated from thestandard and test sample, which remain unbound.

Sandwich assays involve the use of two antibodies, each capable ofbinding to a different immunogenic portion, or epitope, or the proteinto be detected. In a sandwich assay, the test sample to be analyzed isbound by a first antibody, which is immobilized on a solid support, andthereafter a second antibody binds to the test sample, thus forming aninsoluble three-part complex. See e.g., U.S. Pat. No. 4,376,110. Thesecond antibody may itself be labeled with a detectable moiety (directsandwich assays) or may be measured using an anti-immunoglobulinantibody that is labeled with a detectable moiety (indirect sandwichassay). For example, one type of sandwich assay is an ELISA assay, inwhich case the detectable moiety is an enzyme.

The antibodies may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹ Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵ S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or more PCATtargets and the affinity value(Kd) is less than 1×10⁸ M.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art.

For immunohistochemistry, the disease tissue sample may be fresh orfrozen or may be embedded in paraffin and fixed with a preservative suchas formalin (see Examples). The fixed or embedded section contains thesample are contacted with a labeled primary antibody and secondaryantibody, wherein the antibody is used to detect the PCAT proteinexpress in situ. The detailed procedure is shown in the Example.

Array:

“Array” refers to an ordered arrangement of at least two transcripts,proteins or peptides, or antibodies on a substrate. At least one of thetranscripts, proteins, or antibodies represents a control or standard,and the other transcript, protein, or antibody is of diagnostic ortherapeutic interest. The arrangement of at least two and up to about40,000 transcripts, proteins, or antibodies on the substrate assuresthat the size and signal intensity of each labeled complex, formedbetween each transcript and at least one nucleic acid, each protein andat least one ligand or antibody, or each antibody and at least oneprotein to which the antibody specifically binds, is individuallydistinguishable.

An “expression profile” is a representation of gene expression in asample. A nucleic acid expression profile is produced using sequencing,hybridization, or amplification technologies using transcripts from asample. A protein expression profile, although time delayed, mirrors thenucleic acid expression profile and is produced using gelelectrophoresis, mass spectrometry, or an array and labeling moieties orantibodies which specifically bind the protein. The nucleic acids,proteins, or antibodies specifically binding the protein may be used insolution or attached to a substrate, and their detection is based onmethods well known in the art.

A substrate includes but not limits to, paper, nylon or other type ofmembrane, filter, chip, glass slide, or any other suitable solidsupport. The invention also provides an array with a cDNA or transcriptencoding PCAT proteins or peptides or fragments thereof, antibodies thatspecifically bind PCAT proteins, peptides or fragments thereof.Preferably, two or more of the nucleic acid molecules (e.g., SEQ ID NOS:289-702), proteins (e.g., SEQ ID NOS: 1-288) or peptides (e.g., SEQ IDNOS: 703-873) are immobilized on a substrate. Specifically, thefollowing targets are selected for targeting purpose:Tissue Factor,GS3786, Na/K ATPase Beta-3, Kunitz Type inhibitor-2 (HAI-2), CD46,CD166, Neutral Amino Acid Transporter (ASCT2), Transglutaminase-2(TGM2), Kunitz Inhibitor-1, ErbB3, Mucin 4, Decay Accelerating Factor(DAF).

The present invention also provides an antibody array. Antibody arrayshave allowed the development of techniques for high-throughput screeningof recombinant antibodies. Such methods use robots to pick and gridbacteria containing antibody genes, and a filter-based ELISA to screenand identify clones that express antibody fragments. Because liquidhandling is eliminated and the clones are arrayed from master stocks,the same antibodies can be spotted multiple times and screened againstmultiple antigens simultaneously. For more information, see de Wildt etal. (2000) Nat Biotechnol 18: 989-94.

The array is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), U.S.Pat. No. 5,807,522, Brown et al., all of which are incorporated hereinin their entirety by reference.

In one embodiment, a nucleic acid array or a microarray, preferablycomposed of a large number of unique, single-stranded nucleic acidsequences, usually either synthetic antisense oligonucleotides orfragments of cDNAs, fixed to a solid support. The oligonucleotides arepreferably about 6-60 nucleotides in length, more preferably 15-30nucleotides in length, and most preferably about 20-25 nucleotides inlength.

In order to produce oligonucleotides to a known sequence for an array,the gene(s) of interest (or an ORF identified from the contigs of thepresent invention) is typically examined using a computer algorithmwhich starts at the 5′ or at the 3′ end of the nucleotide sequence.Typical algorithms will then identify oligomers of defined length thatare unique to the gene, have a GC content within a range suitable forhybridization, and lack predicted secondary structure that may interferewith hybridization. In certain situations it may be appropriate to usepairs of oligonucleotides on an array. The “pairs” will be identical,except for one nucleotide that preferably is located in the center ofthe sequence. The second oligonucleotide in the pair (mismatched by one)serves as a control. The number of oligonucleotide pairs may range fromtwo to one million. The oligomers are synthesized at designated areas ona substrate using a light-directed chemical process, wherein thesubstrate may be paper, nylon or other type of membrane, filter, chip,glass slide or any other suitable solid support as described above.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/251116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference.

A gene expression profile comprises the expression of a plurality oftranscripts as measured by hybridization with a sample. The transcriptsof the invention may be used as elements on an array to produce a geneexpression profile. In one embodiment, the array is used to diagnose ormonitor the progression of disease. Researchers can assess and catalogthe differences in gene expression between healthy and diseased tissuesor cells.

For example, the transcript or probe may be labeled by standard methodsand added to a biological sample from a patient under conditions for theformation of hybridization complexes. After an incubation period, thesample is washed and the amount of label (or signal) associated withhybridization complexes, is quantified and compared with a standardvalue. If complex formation in the patient sample is significantlyaltered (higher or lower) in comparison to either a normal or diseasestandard, then differential expression indicates the presence of adisorder.

In order to provide standards for establishing differential expression,normal and disease expression profiles are established. This isaccomplished by combining a sample taken from normal subjects, eitheranimal or human or nonmammal, with a transcript under conditions forhybridization to occur. Standard hybridization complexes may bequantified by comparing the values obtained using normal subjects withvalues from an experiment in which a known amount of a purified sequenceis used. Standard values obtained in this manner may be compared withvalues obtained from samples from patients who were diagnosed with aparticular condition, disease, or disorder. Deviation from standardvalues toward those associated with a particular disorder is used todiagnose that disorder.

By analyzing changes in patterns of gene/protein expression, disease canbe diagnosed at earlier stages before the patient is symptomatic. Theinvention can be used to formulate a prognosis and to design a treatmentregimen. The invention can also be used to monitor the efficacy oftreatment. For treatments with known side effects, the array is employedto improve the treatment regimen. A dosage is established that causes achange in genetic expression patterns indicative of successfultreatment. Expression patterns associated with the onset of undesirableside effects are avoided.

In another embodiment, animal models which mimic a human disease can beused to characterize expression profiles associated with a particularcondition, disease, or disorder; or treatment of the condition, disease,or disorder. Novel treatment regimens may be tested in these animalmodels using arrays to establish and then follow expression profilesover time. In addition, arrays may be used with cell cultures or tissuesremoved from animal models to rapidly screen large numbers of candidatedrug molecules, looking for ones that produce an expression profilesimilar to those of known therapeutic drugs, with the expectation thatmolecules with the same expression profile will likely have similartherapeutic effects. Thus, the invention provides the means to rapidlydetermine the molecular mode of action of a drug.

Such assays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies or in clinical trials orto monitor the treatment of an individual patient. Once the presence ofa condition is established and a treatment protocol is initiated,diagnostic assays may be repeated on a regular basis to determine if thelevel of expression in the patient begins to approximate that, which isobserved in a normal subject. The results obtained from successiveassays may be used to show the efficacy of treatment over a periodranging from several days to years.

In one embodiment, the detected targets comprise, consist essentially ofor consist of combinations of PCAT proteins or nucleic acids encodingsuch proteins. The combinations are either 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 proteins(SEQ ID NOS: 1-288), nucleic acids (SEQ ID NOS: 289-702) encoding suchproteins, or peptides (SEQ ID NOS: 703-873).

In one embodiment, the combinations of the protein or nucleic acidtargets for detection of a pancreatic cancer comprise targets selectedfrom a group consisting of Tissue Factor, GS3786, Na/K ATPase Beta-3,Kunitz Type inhibitor-1(HAI-1), Kunitz Type inhibitor-2 (HAI-2), CD46,CD166, Neutral Amino Acid Transporter (ASCT2), Transglutaminase-2(TGM2), Kunitz Inhibitor-1, ErbB3, Mucin 4, Decay Accelerating Factor(DAF), wherein their corresponding sequences are listed in Table 2.

In another embodiment, the detection targets are combination of all thetargets of Tissue Factor, GS3786, Na/K ATPase Beta-3, Kunitz Typeinhibitor-1 (HAI-1), Kunitz Type inhibitor-2 (HAI-2), CD46, CD166,Neutral Amino Acid Transporter (ASCT2), Transglutaminase-2 (TGM2),Kunitz Inhibitor-1, ErbB3, Mucin 4, Decay Accelerating Factor (DAF).

In yet another embodiment, the invention provides a compositioncomprising a plurality of PCAT nucleic acid sequences for use indetecting the differential expression of genes in a disease state,wherein said plurality of nucleic acid sequences comprise two or moresequences of SEQ ID NOS: 289-702, or all the sequences of SEQ ID NOS:289-702, or the complete complements thereof. Further, the nucleic acidsequences are immobilized on a substrate, and the said nucleic acidsequences are hybridizable elements on a microarray.

In yet another embodiment, the invention provides a compositioncomprising a plurality of PCAT proteins or peptides for use in detectingthe differential expression of proteins in a disease state wherein saidplurality of protein sequences comprise two or more sequences of SEQ IDNOS: 1-288 or all sequence of SEQ ID NOS: 1-288; and wherein saidplurality of peptide sequences comprise two or more sequences of SEQ IDNOS: 703-873 or all sequence of SEQ ID NOS: 703-873.

In yet another embodiment, the compositions can be used in diagnosing ormonitoring the treatment of pancreatic cancer

Treatment

The following terms, as used in the present specification and claims,are intended to have the meaning as defined below, unless indicatedotherwise.

“Treat,” “treating” or “treatment” of a disease includes:

(1) inhibiting the disease, i.e., arresting or reducing the developmentof the disease or its clinical symptoms, or (2) relieving the disease,i.e., causing regression of the disease or its clinical symptoms.

A “therapeutically effective amount” means the amount of an agent that,when administered to a subject for treating a disease, is sufficient toeffect such treatment for the disease. The “therapeutically effectiveamount” will vary depending on the agent, the disease and its severityand the age, weight, etc., of the subject to be treated.

A “panceratic disease” includes pancreatic cancer, pancreatic tumor(exocrine or endocrine), pancreatic cysts, acute pancreatitis, chronicpancreatitis, diabetes (type I and II) as well as pancreatic trauma,preferably pancreatic cancer.

A “cancer” is epithelial-cell related cancers include but not limited topancreatic, lung, colon, prostate, ovarian, breast, bladder renal,hepatocellular, pharyngeal and gastric cancers.

The present invention provides an application of treatment by usingantibody, immunogenic peptides as well as other PCAT agonists orantagonists.

PCATs are proteins differentially expressed in the pancreatic diseasescell lines or tissues. The proteins are either cell surface proteins orcytosolic proteins (see the list in Table 2). These proteins areassociated with the diseases especially pancreatic diseases,particularly pancreatic cancer; thus, they serve as candidate targetsfor the treatment of the diseases.

In one embodiment, when decreased expression or activity of the proteinis desired, an inhibitor, antagonist, antibody and the like or apharmaceutical agent containing one or more of these molecules may bedelivered. Such delivery may be effected by methods well known in theart and may include delivery by an antibody specifically targeted to theprotein. Neutralizing antibodies, which inhibit dimer formation, aregenerally preferred for therapeutic use.

In another embodiment, when increased expression or activity of theprotein is desired, the protein, an agonist, an enhancer and the like ora pharmaceutical agent containing one or more of these molecules may bedelivered. Such delivery may be effected by methods well known in theart and may include delivery of a pharmaceutical agent by an antibodyspecifically targeted to the protein.

Any of the transcripts, complementary molecules, or fragments thereof,proteins or portions thereof, vectors delivering these nucleic acidmolecules or expressing the proteins, and their ligands may beadministered in combination with other therapeutic agents. Selection ofthe agents for use in combination therapy may be made by one of ordinaryskill in the art according to conventional pharmaceutical principles. Acombination of therapeutic agents may act synergistically to affecttreatment of a particular disorder at a lower dosage of each agent.

Antibody Therapy

The antibody of the present invention can be used for therapeuticreasons. It is contemplated that the antibody of the present inventionmay be used to treat a mammal, preferably a human with pancreaticdiseases.

In general, the antibodies are also useful for inhibiting proteinfunction, for example, blocking the binding of the PCAT protein orpeptide to a binding partner such as a substrate. These uses can also beapplied in a therapeutic context in which treatment involves inhibitingthe protein's function. An antibody can be used, for example, to blockbinding, thus modulating (agonizing or antagonizing) the peptidesactivity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated within a cell or cell membrane. The function blocking assaysare provided in detail in the Examples. Other evidence is provided inU.S. Pat. No. 6,207,152, and U.S. Patnet No. 6,387,371.

The antibodies of present invention can also be used as means ofenhancing the immune response. The antibodies can be administered inamounts similar to those used for other therapeutic administrations ofantibody. For example, pooled gamma globulin is administered at a rangeof about 1 mg to about 100 mg per patient. Thus, antibodies reactivewith the protein or peptides of PCAT can be passively administered aloneor in conjunction with other anti-cancer therapies to a mammalafflicted-with pancreatic diseases or cancer. Examples of anti-cancertherapies include, but are not limited to, chemotherapy, radiationtherapy, adoptive immunotherapy therapy with TIL (Tumor InfiltrationLymphocytes).

The selection of an antibody subclass for therapy will depend upon thenature of the disease tumor antigen. For example, an IgM may bepreferred in situations where the antigen is highly specific for thediseased target and rarely occurs on normal cells. However, where thedisease-associated antigen is also expressed in normal tissues, althoughat much lower levels, the IgG subclass may be preferred for thefollowing reason: since the binding of at least two IgG molecules inclose proximity is required to activate complement, less complementmediated damage may occur in the normal tissues which express smalleramounts of the antigen and, therefore, bind fewer IgG antibodymolecules. Furthermore, IgG molecules by being smaller may be more ablethan IgM molecules to localize to the diseased tissue.

The mechanism for antibody therapy is that the therapeutic antibodyrecognizes a cell surface protein or a cytosolic protein that isoverexpressed in diseased cells. By NK cell or complement activation,conjugation of the antibody with an immunotoxin or radiolabel, theinteraction can abrogate ligand/receptor interaction or activation ofapoptosis.

The potential mechanisms of antibody-mediated cytotoxicity of diseasedcells are phagocyte (antibody dependent cellular cytotoxicity (ADCC))(see Example), complement (Complement-mediated cytotoxicity (CMC)) (seeExample), naked antibody (receptor cross-linking apoptosis and growthfactor inhibition), or targeted payload labeled with radionuclide orimmunotoxins or immunochemotherapeutics.

In one embodiment, the antibody is administered to a nonhuman mammal forthe purposes of obtaining preclinical data, for example. Exemplarynonhuman mammals to be treated include nonhuman primates, dogs, cats,rodents and other mammals in which preclinical studies are performed.Such mammals may be established animal models for a disease to betreated with the antibody or may be used to study toxicity of theantibody of interest. In each of these embodiments, dose escalationstudies may be performed on the mammal.

The antibody is administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local immunosuppressive treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody variant issuitably administered by pulse infusion, particularly with decliningdoses of the antibody variant. Preferably the dosing is given byinjections, most preferably intravenous or subcutaneous injections,depending in part on whether the administration is brief or chronic.

For the prevention or treatment of a disease, the appropriate dosage ofthe antibody will depend on the type of disease to be treated, theseverity and the course of the disease, whether the antibody isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician.

Depending on the type and severity of the disease, about 1 .mu.g/kg to150 mg/kg (e.g., 0.1-20 mg/kg) of antibody is an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

The antibody composition will be formulated, dosed and administered in amanner consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.

The therapeutically effective amount of the antibody to be administeredwill be governed by such considerations, and is the minimum amountnecessary to prevent, ameliorate, or treat a disease or disorder. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question.

Antibodies of the present invention may also be used as therapeuticreagents, to diminish or eliminate cancer or tumors. For example, theantibodies may be used on their own (for instance, to inhibitmetastases) or coupled to one or more therapeutic agents. Suitableagents in this regard include radionuclides, differentiation inducers,drugs, toxins, and derivatives thereof. Preferred radionuclides include⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and .²¹²Bi. Preferred drugsinclude methotrexate, and pyrimidine and purine analogs. Preferreddifferentiation inducers include phorbol esters and butyric acid.Preferred toxins include ricin, abrin, diptheria toxin, cholera toxin,gelonin, Pseudomonas exotoxin, Shigella toxin, and pokeweed antiviralprotein

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be affected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g. U.S. Pat. No.4,671,958, to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No. 4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways as described above.

While it is possible for the immunogen to be administered in a pure orsubstantially pure form, it is preferable to present it as apharmaceutical composition, formulation or preparation with a carrier.

The formulations of the present invention, both for veterinary and forhuman use, comprise an immunogen as described above, together with oneor more pharmaceutically acceptable carriers and, optionally, othertherapeutic ingredients. The carrier(s) must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethod well-known in the pharmaceutical art.

Suitable pharmaceutical carriers include proteins such as albumins(e.g., U.S. Pat. No. 4,507,234, to Kato et al.), peptides andpolysaccharides such as aminodextran (e.g., U.S. Pat. No. 4,699,784 toShih et al.); or water. A carrier may also bear an agent by noncovalentbonding or by encapsulation, such as within a liposome vesicle (e.g.,U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriers specific forradionuclide agents include radiohalogenated small molecules andchelating compounds. For example, U.S. Pat. No. 4,735,792 disclosesrepresentative radiohalogenated small molecules and their synthesis. Aradionuclide chelate may be formed from chelating compounds that includethose containing nitrogen and sulfur atoms as the donor atoms forbinding the metal, metal oxide, radionuclide. For example, U.S. Pat. No.4,673,562, to Davison et al. discloses representative chelatingcompounds and their synthesis.

All methods include the step of bringing into association the activeingredient with the carrier, which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations suitable for intravenous intramuscular, subcutaneous, orintraperitoneal administration conveniently comprise sterile aqueoussolutions of the active ingredient with solutions, which are preferablyisotonic with the blood of the recipient. Such formulations may beconveniently prepared by dissolving solid active ingredient in watercontaining physiologically compatible substances such as sodium chloride(e.g. 0.1-2.0M), glycine, and the like, and having a buffered pHcompatible with physiological conditions to produce an aqueous solution,and rendering said solution sterile. These may be present in unit ormulti-dose containers, for example, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer.Illustrative stabilizers are polyethylene glycol, proteins, saccharides,amino acids, inorganic acids, and organic acids, which may be usedeither on their own or as admixtures. These stabilizers are preferablyincorporated in an amount of 0.11-10,000 parts by weight per part byweight of immunogen. If two or more stabilizers are to be used, theirtotal amount is preferably within the range specified above. Thesestabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of 0.1-3.0 osmoles, preferably inthe range of 0.8-1.2. The pH of the aqueous solution is adjusted to bewithin the range of 5.0-9.0, preferably within the range of 6-8. Informulating the antibody of the present invention, anti-adsorption agentmay be used.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achievedthrough the use of polymer to complex or absorb the proteins or theirderivatives. The controlled delivery may be exercised by selectingappropriate macromolecules (for example polyester, polyamino acids,polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, or protamine sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled-release preparations is to incorporate the PCATantibody into particles of a polymeric material such as polyesters,polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetatecopolymers. Alternatively, instead of incorporating these agents intopolymeric particles, it is possible to entrap these materials inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly(methylmethacylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combinedwith typical carriers, such as lactose, sucrose, starch, talc magnesiumstearate, crystalline cellulose, methyl cellulose, carboxymethylcellulose, glycerin, sodium alginate or gum arabic among others.

The therapeutic antibody may be supplied in the form of a kit, alone, orin the form of a pharmaceutical composition as described above.

Other Immunotherapy

The PCAT proteins, peptides, or fragments thereof of this invention arealso intended for use in producing antiserum designed for pre- orpost-disease prophylaxis. Here the protein, peptides or fragmentthereof, is formulated with a suitable adjuvant and administered byinjection to human volunteers, according to known methods for producinghuman antisera. Antibody response to the injected proteins is monitored,during a several-week period following immunization, by periodic serumsampling to detect the presence of antiserum antibodies, using animmunoassay as described herein.

The antiserum from immunized individuals may be administered as aprophylactic measure for individuals who are at risk of developingpancreatic diseases or cancer. The antiserum is also useful in treatingan individual afflicted with pancreatic diseases or cancer forpost-disease prophylaxis.

Alternatively, peptides derived form the PCAT protein sequence may bemodified to increase their immunogenicity by enhancing binding of thepeptide to the MHC molecules in which the peptide is presented. Thepeptide or modified peptide may be conjugated to a carrier molecule toenhance the antigenicity of the peptide. Examples of carrier molecules,include, but are not limited to, human albumin, bovine albumin,lipoprotein and keyhole limpet hemo-cyanin (“Basic and Clinicalimmunology” (1991) Stites, D. P. and Terr A. I. (eds) Appleton andLange, Norwalk Conn., San Mateo, Calif.).

An “immunogenic peptide” is a peptide, which comprises anallele-specific motif such that the peptide will bind the MHC allele(HLA in human) and be capable of inducing a CTL (cytoxic T-lymphocytes)response. Thus, immunogenic peptides are capable of binding to anappropriate class I or II MHC molecule and inducing a cytotoxic T cellor T helper cell response against the antigen from which the immunogenicpeptide is derived.

Alternatively, amino acid sequence variants of the peptide can beprepared by mutations in the DNA, which encodes the peptide, or bypeptide synthesis.

At the genetic level, these variants ordinarily are prepared bysite-directed mutagenesis of nucleotides in the DNA encoding the peptidemolecule, thereby producing DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture. The variants typicallyexhibit the same qualitative biological activity as the nonvariantpeptide.

T-lymphocytes recognize antigen in association with Class I or Class IIMHC molecules in the form of a peptide fragment bound to an MHCmolecule. The degree of peptide binding to a given MHC allele is basedon amino acids at particular positions within the peptide-(Parker et al.(1992) Journal of Immunology 149: 3580; Kubo, et al. (1994) Journal ofImmunology 52: 3913-3924; Ruppert J. et al. (1993) Cell 74: 929-937;Falk et al. (1991) Nature 351: 290-296). The peptides of the presentinvention are useful as an epitope for immunogenic response (see moredetailed description below).

In human, MHC is called HLA, wherein class I molecules are encoded bythe HLA-A, B, and C loci. HLA-A and B antigens are expressed at the cellsurface at approximately equal densities, whereas the expression ofHLA-C is significantly lower (about 10-fold lower). Each of these locihas a number of alleles. MHC class II molecules are encoded by threepairs of MHC II alpha- and beta-chain genes, called HLA DR, -DP, and -DQin human. In many haplotypes the HLA-DR cluster contains an extrabeta-chain gene whose product can pair with the DR alpha chain. Each MHCclass I and II molecule binds a different rage of peptides. The presentof several loci means that any one individual is equipped to present amuch broader ranger of different peptides than if only one MHC proteinof each class were expressed at the cell surface. The peptide bindingmotifs of the present invention are designed to be specific for eachallelic subtype.

The peptides of the present invention are used for treatment of thepancreatic diseases. Treatment involves administration of the protectivecomposition after the appearance of the disease.

The present invention is also applied to prevent and suppress thedisease. It is not always possible to distinguish between “preventing”and “suppressing” since the ultimate inductive event or events may beunknown, latent, or the patient is not ascertained until well after theoccurrence of the event or events. Therefore, it is common to use theterm “prophylaxis” as distinct from “treatment” to encompass both“preventing” and “suppressing” as defined herein. The term “protection,”as used herein, is meant to include “prophylaxis.”

The peptides are used for treating T cell-mediated pathology. The term“T cell-mediated pathology” refers to any condition in which aninappropriate T cell response is a component of the pathology. The termis intended to encompass both T cell mediated pancreatic diseases anddiseases resulting from unregulated clonal T cell replication.

Therefore, the present invention relates to peptides or modifiedpeptides derived from the protein sequences of the PCAT proteins thatdifferentially expressed in the pancreatic diseases. By way of example,modification may include substitution, deletion or addition of an aminoacid in the given immunogenic peptide sequence or mutation of existingamino acids within the given immunogenic peptide sequence, orderivatization of existing amino acids within the given immunogenicpeptide sequence. Any amino acid comprising the immunogenic peptidesequence may be modified in accordance with this invention. In oneaspect, at least one amino acid is substituted or replaced within thegiven immunogenic peptide sequence. Any amino acid may be used tosubstitute or replace a given amino acid within the immunogenic peptidesequence. Modified peptides are intended to include any immunogenicpeptide obtained from differentially expressed proteins, which has beenmodified and exhibits enhanced binding to the MHC molecule with which itassociates when presented to the T-cell. These modified peptides may besynthetically or recombinantly produced by conventional methods.

In another embodiment, the peptides of the present invention comprise,or consisting sequences of about 5-8, 8-10, 10-15 or 15-30 amino acidswhich are immunogenic, that is, capable of inducing an immune responsewhen injected into a subject.

The recombinant or natural protein, peptides, or fragment thereof ofPCAT, or modified peptides, may be used as a vaccine eitherprophylactically or therapeutically. When provided prophylactically thevaccine is provided in advance of any evidence of pancreatic diseases,particularly, cancer. The prophylactic administration of the pancreaticdisease vaccine should serve to prevent or attenuate pancreaticdiseases, preferably cancer, in a mammal.

Preparation of vaccine is using recombinant protein or peptideexpression vectors comprising all or part of nucleic acid sequence ofPCAT proteins encoding peptides. Examples of vectors that may be used inthe aforementioned vaccines include, but are not limited to, defectiveretroviral vectors, adenoviral vectors vaccinia viral vectors, fowl poxviral vectors, or other viral vectors (Mulligan, R. C., (1993) Science260: 926-932). The viral vectors carrying all or part of nucleicsequence of SEQ ID NOS: 289-702 can be introduced into a mammal eitherprior to any evidence of pancreatic diseases or to mediate regression ofthe disease in a mammal afflicted with pancreatic diseases. Examples ofmethods for administering the viral vector into the mammals include, butare not limited to, exposure of cells to the virus ex vivo, or injectionof the retrovirus or a producer cell line of the virus into the affectedtissue or intravenous administration of the virus. Alternatively theviral vector carrying all or part of the PCAT nucleic acid sequence thatencode peptides may be administered locally by direct injection into thecancer lesion or topical application in a pharmaceutically acceptablecarrier. The quantity of viral vector, carrying all or part of the PCATnucleic acid sequence, to be administered is based on the titer of virusparticles. A preferred range of the immunogen to be administered may beabout 106 to about 1011 virus particles per mammal, preferably a human.After immunization the efficacy of the vaccine can be assessed byproduction of antibodies or immune cells that recognize the antigen, asassessed by specific lytic activity or specific cytokine production orby tumor regression. One skilled in the art would know the conventionalmethods to assess the aforementioned parameters. If the mammal to beimmunized is already afflicted with cancer, the vaccine can beadministered in conjunction with other therapeutic treatments. Examplesof other therapeutic treatments includes, but are not limited to,adoptive T cell immunotherapy, coadministration of cytokines or othertherapeutic drugs for cancer.

Alternatively all or parts thereof of a substantially or partiallypurified the PCAT proteins or their peptides may be administered as avaccine in a pharmaceutically acceptable carrier. Ranges of the proteinthat may be administered are about 0.001 to about 100 mg per patient,preferred doses are about 0.01 to about 100 mg per patient. In apreferred embodiment, the peptides or modified peptides thereof isadministered therapeutically or prophylactically to a mammal in need ofsuch treatment. The peptide may be synthetically or recombinantlyproduced. Immunization is repeated as necessary, until a sufficienttiter of anti-immunogen antibody or immune cells has been obtained.

In yet another alternative embodiment a viral vector, such as aretroviral vector, can be introduced into mammalian cells. Examples ofmammalian cells into which the retroviral vector can be introducedinclude, but are not limited to, primary mammalian cultures orcontinuous mammalian cultures, COS cells, NIH3T3, or 293 cells (ATTC#CRL 1573), dendritic cells. The means by which the vector carrying thegene may be introduced into a cell includes, but is not limited to,microinjection, electroporation, transfection or transfection using DEAEdextran, lipofection, calcium phosphate or other procedures known to oneskilled in the art (Sambrook et al. (EDS) (2001) in “Molecular Cloning.A laboratory manual”, Cold Spring Harbor Press Plainview, N.Y.).

The vaccine formulation of the present invention comprises an immunogenthat induces an immune response directed against the cancer associatedantigens such as the PCATs, and in nonhuman primates and finally inhumans. The safety of the immunization procedures is determined bylooking for the effect of immunization on the general health of theimmunized animal (weight change, fever, appetite behavior etc.) andlooking for pathological changes on autopsies. After initial testing inanimals, cancer patients can be tested. Conventional methods would beused to evaluate the immune response of the patient to determine theefficiency of the vaccine.

Measurement of candidate disease tumor antigen or vaccine expression inpatients is the first step of the present invention. Subsequent stepswill focus on measuring immune responses to these candidate antigens orvaccine. Sera from disease patients, particularly cancer patients, andhealthy donors will be screened for antibodies to the candidate antigensas well as for levels of circulating tumor derived antigens. antigen.The vaccine formulations may be evaluated first in animal models,initially rodents

In one embodiment mammals, preferably human, at high risk for pancreaticdiseases, particularly cancer, are prophylactically treated with thevaccines of this invention. Examples of such mammals include, but arenot limited to, humans with a family history of pancreatic diseases,humans with a history of pancreatic diseases, particular cancer, orhumans afflicted with pancreatic cancer previously resected andtherefore at risk for reoccurrence. When provided therapeutically, thevaccine is provided to enhance the patient's own immune response to thediseased antigen present on the pancreatic diseases or advanced stage ofpancreatic diseases. The vaccine, which acts as an immunogen, may be acell, cell lysate from cells transfected with a recombinant expressionvector, cell lysates from cells transfected with a recombinantexpression vector, or a culture supernatant containing the expressedprotein. Alternatively, the immunogen is a partially or substantiallypurified recombinant protein, peptide or analog thereof or modifiedpeptides or analogs thereof. The proteins or peptides may be conjugatedwith lipoprotein or administered in liposomal form or with adjuvant.

While it is possible for the immunogen to be administered in a pure orsubstantially pure form, it is preferable to present it as apharmaceutical composition, formulation or preparation. The formulationsof the present invention are described in the previous section.

Vaccination can be conducted by conventional methods. For example, theimmunogen can be used in a suitable diluent such as saline or water, orcomplete or incomplete adjuvants. Further, the immunogen may or may notbe bound to a carrier to make the protein immunogenic. Examples of suchcarrier molecules include but are not limited to bovine serum albumin(BSA), keyhole limpet hemocyanin (KLH), tetanus toxoid, and the like.The immunogen also may be coupled with lipoproteins or administered inliposomal form or with adjuvants. The immunogen can be administered byany route-appropriate for antibody production such as intravenous,intraperitoneal, intramuscular, subcutaneous, and the like. Theimmunogen may be administered once or at periodic intervals until asignificant titer of anti-PCAT immune cells or anti-PCAT antibody isproduced. The presence of anti-PCAT immune cells may be assessed bymeasuring the frequency of precursor CTL (cytoxic T-lymphocytes) againstPCAT antigen prior to and after immunization by a CTL precursor analysisassay (Coulie, P. et al., (1992) International Journal Of Cancer 50:289-297). The antibody may be detected in the serum using theimmunoassay described above.

The safety of the immunization procedures is determined by looking forthe effect of immunization on the general health of the immunizedanimal. (weight change, fever, appetite behavior etc.) and looking forpathological changes on autopsies. After initial testing in animals,pancreatic diseases in patients can be tested. Conventional methodswould be used to evaluate the immune response of the patient todetermine the efficiency of the vaccine.

In yet another embodiment of this invention all, part, or parts of thePCAT proteins or peptides or fragments thereof, or modified peptides,may be exposed to dendritic cells cultured in vitro. The cultureddendritic cells provide a means of producing T-cell dependent antigenscomprised of dendritic cell modified antigen or dendritic cells pulsedwith antigen, in which the antigen is processed and expressed on theantigen activated dendritic cell. The PCAT antigen activated dendriticcells or processed dendritic cell antigens may be used as immunogens forvaccines or for the treatment of pancreatic diseases, particularlypancreatic cancer. The dendritic cells should be exposed to antigen forsufficient time to allow the antigens to be internalized and presentedon the dendritic cells surface. The resulting dendritic cells or thedendritic cell process antigens can than be administered to anindividual in need of therapy. Such methods are described in Steinman etal. (WO93/208185) and in Banchereau et al. (EPO Application 0563485A1).

In yet another aspect of this invention T-cells isolated fromindividuals can be exposed to the PCAT proteins, peptides or fragmentsthereof, or modified peptides in vitro and then administered to apatient in need of such treatment in a therapeutically effective amount.Examples of where T-lymphocytes can be isolated include but are notlimited to, peripheral blood cells lymphocytes (PBL), lymph nodes, ortumor infiltrating lymphocytes (TIL). Such lymphocytes can be isolatedfrom the individual to be treated or from a donor by methods known inthe art and cultured in vitro (Kawakami, Y. et al. (1989) J. Immunol.142: 2453-3461). Lymphocytes are cultured in media such as RPMI or RPMI1640 or AIM V for 1-10 weeks. Viability is assessed by trypan blue dyeexclusion assay. Examples of how these sensitized T-cells can beadministered to the mammal include but are not limited to,intravenously, intraperitoneally or intralesionally. Parameters that maybe assessed to determine the efficacy of these sensitized T-lymphocytesinclude, but are not limited to, production of immune cells in themammal being treated or tumor regression. Conventional methods are usedto assess these parameters. Such treatment can be given in conjunctionwith cytokines or gene modified cells (Rosenberg, S. A. et al. (1992)Human Gene Therapy, 3: 75-90; Rosenberg, S. A. et al. (1992) Human GeneTherapy, 3: 57-73).

The present invention is further described by the following example. Theexample is provided solely to illustrate the invention by reference tospecific embodiments. This exemplification, while illustrating certainaspects of the invention, does not offer the limitations or circumscribethe scope of the disclosed invention.

All examples outlined here were carried out using standard techniques,which are well known and routine to those of skill in the art. Routinemolecular biology techniques of the following example can be carried outas described in standard laboratory manuals, such as Sambrook et al.,Molecular Cloning: A laboratory Manual, 3^(rd) Ed.; Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (2001).

WORKING EXAMPLES 1. Pancreatic Cell Line Model System

Analysis of gene expression in various pancreatic cancer cell lines aswell as pancreatic duct epithelial tissue has shown that the cell lineHs766T correlates well with normal tissue. For this reason, this cellline is reported in the literature as being a good surrogate for normaltissue in analyses of differential expression between pancreaticadenomcarcinoma (and derived tumor lines) and normal tissue (orsurrogate, Hs766T). The model system employed here involves the use ofHs766T as a “normal” reference to which cell surface expression in tumorderived cell lines is compared. These differentials or candidates arethen validated in tissues, pancreatic cancer and normal pancreas, toconfirm that they are differentially expressed between these tissues aswell as within the cell line model system. Details of the pancreatictumor lines that were used for this study, as well as the pancreaticline Hs766T are provided below.

TABLE 3 Cell Lines and Media Non- Fetal ATCC Base essential SodiumSodium Bovine Cell line Reference medium Glutamine amino acids CarbonatePyruvate Hepes Serum Panc-1 CRL-1469 DMEM 2 mM 1% (w/v) 0.1% (w/v) 1 mM10% (v/v) Hs766t HTB-134 DMEM 2 mM 1% (w/v) 0.1% (w/v) 1 mM 10% (v/v)SU.86.86 CRL-1837 DMEM 2 mM 1% (w/v) 0.1% (w/v) 1 mM 10% (v/v) AsPC1CRL-1682 RPMI 2 mM 1% (w/v) 0.1% (w/v) 1 mM 10 mM 20% (v/v) HPAF IICRL-1997 DMEM 2 mM 1% (w/v) 0.1% (w/v) 1 mM 10% (v/v) HPAC CRL-2119 DMEM2 mM 1% (w/v) 0.1% (w/v) 1 mM 10% (v/v) Mia-Paca-2 CRL-1420 DMEM 2 mM 1%(w/v) 0.1% (w/v) 1 mM 10% (v/v) Mpanc-96 CRL-2380 RPMI 2 mM 1% (w/v)0.1% (w/v) 1 mM 10 mM 10% (v/v) BxPC-3 CRL-1687 RPMI 2 mM 1% (w/v) 0.1%(w/v) 1 mM 10 mM 10% (v/v) Capan-2 HTB-80 DMEM 2 mM 1% (w/v) 0.1% (w/v)1 mM 10% (v/v)Pancreatic Cancer Cell Line Culture

Cell lines were grown in a culturing medium that is supplemented asnecessary with growth factors and serum (as described in Table 3).Cultures were established from frozen stocks in which the cells weresuspended in a freezing medium (cell culture medium with 10% DMSO [v/v])and flash frozen in liquid nitrogen. Frozen stocks prepared in this waywere stored in the liquid nitrogen vapour. Cell cultures wereestablished by rapidly thawing frozen stocks at 37° C. Thawed stockcultures were slowly transferred to a culture vessel containing a largevolume of culture medium that was supplemented. For maintenance ofculture, cells were seeded at 1×10⁵ cells/per ml in medium and incubatedat 37° C. until confluence of cells in the culture vessel exceeds 50% byarea. At this time, cells were harvested from the culture vessel usingenzymes or EDTA where necessary. The density of harvested, viable cellswas estimated by hemocytometry and the culture reseeded as above. Apassage of this nature was repeated no more than 25 times at which pointthe culture was destroyed and reestablished from frozen stocks asdescribed above.

For the analyses of cell surface protein expression in cultured celllines cells were grown as described above. At a period 24 h prior to theexperiment, the cell line was passaged as described above. This yieldedcell densities that were <50% confluent and growing exponentially.Typically, triplicate analyses of differential expression were performedfor each line relative to Hs766T for the purpose of identifyingstatistically significant reproducible differentially expressedproteins.

2. Cloning and Expression of Target Proteins

cDNA Retrieval

Peptide sequences were searched by BlastP against the Celera DiscoverySystem (CDS) and public database to identify the correspondingfull-length open reading frames (ORFs). Each ORF sequence was thensearched by BlastN against the Celera in-house human cDNA clonecollection. For each sequence of interest, up to three clones werepulled and streaked onto LB/Ampicillin (100 ug/ml) plates. Plasmid DNAwas isolated using Qiagen spin mini-prep kit and verified by restrictiondigest. Subsequently, the isolated plasmid DNA was sequence verifiedagainst the ORF reference sequence. Sequencing reactions were carriedout using Applied Biosystems BigDye Terminator kit followed by ethanolprecipitation. Sequence data was collected using the Applied Biosystems3100 Genetic Analyzer and analyzed by alignment to the referencesequence using the Clone Manager alignment tool.

PCR

PCR primers were designed to amplify the full-length ORF as well as anyregions of the ORF that were interest for expression (antigenic orhydrophilic regions as determined by the Clone Manager sequence analysistool). Primers also contained 5′ and 3′ overhangs to facilitate cloning(see below). PCR reactions contained 2.5 units Platinum Taq DNAPolymerase High Fidelity (Invitrogen), 50 ng cDNA plasmid template, 1 uMforward and reverse primers, 800 uM dNTP cocktail (Applied Biosystems)and 2 mM MgSO₄. After 20-30 cycles (94° C. for 30 seconds, 55° C. for 1minutes and 73° C. for 2 minutes), product was verified and quantitatedby agarose gel electrophoresis.

Construction of Entry Clones

PCR products were cloned into an entry vector for use with the Gatewayrecombination based cloning system (Invitrogen). These vectors includedpDonr221, pDonr201, pEntr/D-TOPO or pEntr/SD/D-TOPO and were used asdescribed in the cloning methods below.

TOPO Cloning into pEntr/D-TOPO or pEntr/SD/D-TOPO

For cloning using this method, the forward PCR primer contained a 5′overhang containing the sequence “CACC”. PCR products were generated asdescribed above and cloned into the entry vector using the InvitrogenTOPO cloning kit. Reactions were typically carried out at roomtemperature for 10 minutes and subsequently transformed into TOP10chemically competent cells (Invitrogen, CA). Candidate clones werepicked, plasmid DNA was prepared using Qiagen spin mini-prep kit andscreened using restriction digest. Inserts were subsequently sequenceverified as described above.

Gateway Cloning into pDonr201 or pDonr221

For cloning using this method, PCR primers contained the followingoverhangs: Forward 5′ overhange:

5′-GGGGACAAGTTTGTAAAAAAGCAGGCTTC-3′ (SEQ ID NO:874) Reverse 5′overhange:

5′-GGGGACCATTTGTACAAGAAAGCTGGGT-3′ (SEQ ID NO:875)

PCR products were generated as described above. ORFs were recombinedinto the entry vector using the Invitrogen Gateway BP Clonase enzymemix. Reactions were typically carried out at 25° C. for 1 hour, treatedwith Proteinase K at 37° C. for 10 minutes and transformed into LibraryEfficiency DH5α chemically competent cells (Invitrogen, CA). Candidateclones were picked, plasmid DNA was prepared using Qiagen spin mini-prepkit and screened using restriction digest. Inserts were subsequentlysequence verified as described above.

Construction of Expression Clones

ORFs were transferred from the entry construct into a series ofexpression vectors using the Gateway LR Clonase enzyme mix. Reactionswere typically carried out for 1 hour at 25° C., treated with ProteinaseK at 37° C. for 10 minutes and subsequently transformed into LibraryEfficiency DH5α chemically competent cells (Invitrogen). Candidateclones were picked, plasmid DNA was prepared using Qiagen spin mini-prepkit and screened using restriction digest. Expression vectors includedbut were not limited to pDest14, pDest15, pDest17, pDest8, pDest10 andpDest20. These vectors allow expression in systems such as E. coli andrecombinant baculovirus. Other vectors not listed here allow expressionin yeast, mammalian cells, or in vitro.

Expression of Recombinant Proteins in E. coli

Constructs were transformed into one or more of the following hoststrains: BL21 SI, BL21 AI, (Invitrogen); Origami B (DE3), Origami B(DE3) pLysS, Rosetta (DE3), Rosetta (DE3) pLysS, Rosetta-Gami (DE3),Rosetta-Gami (DE3) pLysS, or Rosetta-Gami B (DE3) pLysS (Novagen). Thetransformants were grown in LB with or without NaCl and with appropriateantibiotics, at temperatures in the range of 20-37° C., with aeration.Expression was induced with the addition of IPTG (0.03-0.3 mM) or NaCl(75-300 mM) when the cells were in mid-log growth. Growth was continuedfor one to 24 hours post-induction. Cells were harvested bycentrifugation in a Sorvall RC-3 C centrifuge in a H6000A rotor for 10minutes at 3000 rpm, at 4° C. Cell pellets were stored at −80° C.

Expression of Recombinant Proteins Using Baculovirus

Recombinant proteins were expressed using baculovirus in Sf21 fall armyworm ovarian cells. Recombinant baculoviruses were prepared using theBac-to-Bac system (Invitrogen) per the manufacturer's instructions.Proteins were expressed on the large scale in Sf900II serum-free medium(Invitrogen) in a 10 L bioreactor tank (27° C., 130 rpm, 50% dissolvedoxygen for 48 hours).

3. Recombinant Protein Purification

Recombinant proteins were purified from E. coli and/or insect cellsusing a variety of standard chromatography methods. Briefly, cells werelysed using sonication pr detergents. The insoluble material waspelleted by centrifugation at 10,000×g for 15 minutes. The supernatantwas applied to an appropriate affinity column, e.g. His-tagged proteinswere separated using a pre-packed chelating sepharose column (Pharmacia)or GST-tagged proteins were separated using a glutathione sepharosecolumn (Pharmacia). After using the affinity column, proteins werefurther separated using various techniques, such as ion exchangechromatography (columns from Pharmacia) to separate on the basis ofelectrical charge or size exclusion chromatography (columns fromTosohaas) to separate on the basis of molecular weight, size and shape.

Expression and purification of the protein are also achieved usingeither a mammalian cell expression system or an insect cell expressionsystem. The pUB6/V5-His vector system (Invitrogen, CA) is used toexpress GSCC in CHO cells. The vector contains the selectable bsd gene,multiple cloning sites, the promoter/enhancer sequence from the humanubiquitin C gene, a C-terminal V5 epitope for antibody detection withanti-V5 antibodies, and a C-terminal polyhistidine (6.times.His)sequence for rapid purification on PROBOND resin (Invitrogen, CA).Transformed cells are selected on media containing blasticidin.

Spodoptera frugiperda (Sf9) insect cells are infected with recombinantAutographica californica nuclear polyhedrosis virus (baculovirus). Thepolyhedrin gene is replaced with the cDNA by homologous recombinationand the polyhedrin promoter drives cDNA transcription. The protein issynthesized as a fusion protein with 6× his, which enables purificationas described above. Purified protein is used in the following activityand to make antibodies

4. Chemical Synthesis of Peptides

Proteins or portions thereof may be produced not only by recombinantmethods, but also by using chemical methods well known in the art. Solidphase peptide synthesis may be carried out in a batchwise or continuousflow process, which sequentially adds .alpha.-amino- and sidechain-protected amino acid residues to an insoluble polymeric supportvia a linker group. A linker group such as methylamine-derivatizedpolyethylene glycol is attached to poly(styrene-co-divinylbenzene) toform the support resin. The amino acid residues are N-a-protected byacid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc(9-fluorenylmethoxycarbonyl). The carboxyl group of the protected aminoacid is coupled to the amine of the linker group to anchor the residueto the solid phase support resin. Trifluoroacetic acid or piperidine areused to remove the protecting group in the case of Boc or Fmoc,respectively. Each additional amino acid is added to the anchoredresidue using a coupling agent or pre-activated amino acid derivative,and the resin is washed. The full length peptide is synthesized bysequential deprotection, coupling of derivitized amino acids, andwashing with dichloromethane and/or N,N-dimethylformamide. The peptideis cleaved between the peptide carboxy terminus and the linker group toyield a peptide acid or amide. (Novabiochem 1997/98 Catalog and PeptideSynthesis Handbook, San Diego Calif. pp. S1-S20). Automated synthesismay also be carried out on machines such as the 431A peptide synthesizer(ABI). A protein or portion thereof may be purified by preparative highperformance liquid chromatography and its composition confirmed by aminoacid analysis or by sequencing (Creighton (1984) Proteins, Structuresand Molecular Properties, W H Freeman, New York N.Y.).

5. Antibody Development

Polyclonal Antibody Preparations:

Polyclonal antibodies against recombinant proteins were raised inrabbits (Green Mountain Antibodies, Burlington, Vt.). Briefly, two NewZealand rabbits were immunized with 0.1 mg of antigen in completeFreund's adjuvant. Subsequent immunizations were carried out using 0.05mg of antigen in incomplete Freund's adjuvant at days 14, 21 and 49.Bleeds were collected and screened for recognition of the antigen bysolid phase ELISA and western blot analysis. The IgG fraction wasseparated by centrifugation at 20,000×g for 20 minutes followed by a 50%ammonium sulfate cut. The pelleted protein was resuspended in 5 mM Trisand separated by ion exchange chromatography. Fractions were pooledbased on IgG content. Antigen-specific antibody was affinity purifiedusing Pierce AminoLink resin coupled to the appropriate antigen.

Isolation of Antibody Fragments Directed Against PCATs from a Library ofscFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstPCAT to which the donor may or may not have been exposed (see e.g., U.S.Pat. No. 5,885,793 incorporated herein by reference in its entirety).

Rescue of the Library: A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 109 E. coli harboring thephagemid are used to inoculate 50 ml of 2× TY containing 1% glucose and100 .mu.g/ml of ampicillin (2.times.TY-AMP-GLU) and grown to an O.D. of0.8 with shaking. Five ml of this culture is used to innoculate 50 ml of2.times.TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III,see PCT publication WO 92/01047) are added and the culture incubated at37° C. for 45 minutes without shaking and then at 37° C. for 45 minuteswith shaking. The culture is centrifuged at 4000 r.p.m. for 10 min. andthe pellet resuspended in 2 liters of 2×TY containing 100 .mu.g/mlampicillin and 50 ug/ml kanamycin and grown overnight. Phage areprepared as described in PCT publication WO 92/01047.

M13 delta gene IIIis prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M113 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2×TY broth containing 100 .mu.g ampicillin/ml and25 .mu.g kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37°C. Phagre particles are purified and concentrated from the culturemedium by two PEG-precipitations (Sambrook et al., 2001), resuspended in2 ml PBS and passed through a 0.45 .mu.m filter (MINISART NML;Sartorius) to give a final concentration of approximately 1013transducing units/ml (ampicillin-resistant clones).

Panning of the Library: Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 .mu.g/ml or 10 .mu.g/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phages are then used to infect 10 ml of mid-log E.coli TG1 by incubating eluted phage with bacteria for 30 minutes at 37°C. The E. coli are then plated on TYE plates containing 1% glucose and100 .mu.g/ml ampicillin. The resulting bacterial library is then rescuedwith delta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders: Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 .mu.g/ml ofthe polypeptide of the present invention in 50 mM bicarbonate pH 9.6.Clones positive in ELISA are further characterized by PCR fingerprinting(see, e.g., PCT publication WO 92/01047) and then by sequencing.

Monoclonal Antibody Generation

i) Materials:

Complete Media No Sera (CMNS) for washing of the myeloma and spleencells; Hybridoma medium CM-HAT{Cell Mab (BD), 10% FBS (or HS); 5% OrigenHCF (hybridoma cloning factor) containing 4 mM L-glutamine andantibiotics} to be used for plating hybridomas after the fusion.

2) Hybridoma medium CM-HT (NO AMINOPTERIN) (Cell Mab (BD), 10% FBS 5%Origen HCF containing 4 mM L-glutamine and antibiotics) to be used forfusion maintenance are stored in the refrigerator at 4-6° C. The fusionsare fed on days 4, 8, and 12, and subsequent passages. Inactivated andpre-filtered commercial Fetal Bovine serum (FBS) or Horse Serum (HS) arethawed and stored in the refrigerator at 4° C. and must be pretested formyeloma growth from single cells.

The L-glutamine (200 mM, 100× solution), which is stored at −20° C.freezer, is thawed and warmed until completely in solution. TheL-glutamin is dispensed into media to supplement growth. L-glutamin isadded to 2 mM for myelomas, and 4 mM for hybridoma media. Further thePenicillin, Streptomycin, Amphotericin (antibacterial-antifungal storedat −20° C.) is thawed and added to Cell Mab Media to 1%.

4) Myeloma growth media is Cell Mab Media (Cell Mab Media, Quantum Yieldfrom BD is stored in the refrigerator at 4° C. in the dark) which areadded L-glntamine to 2 mM and antibiotic/antimycotic solution to 1% andis called CMNS.

5) 1 bottle of PEG 1500 in Hepes (Roche, N.J.)

6) 8-Azaguanine is stored as the dried powder supplied by SIGMA at −700°C. until needed. Reconstitute 1 vial/500 ml of media and add entirecontents to 500 ml media (eg. 2 vials/liter).

7) Myeloma Media is CM which has 10% FBS (or HS) and 8-Aza (1×) storedin the refrigerator at 4° C.

8) Clonal cell medium D (Stemcell, Vancouver) contains HAT and methylcellulose for semi-solid direct cloning from the fusion.

9) Hybridoma supplements HT [hypoxanthine, thymidine] are to be used inmedium for the section of hybridomas and maintenace of hybridomasthrough the cloning stages respectively.

10) Origen HCF can be obtained directly from Igen and is a cellsupernatant produced from a macrophage-like cell-line. It can be thawedand aliqouted to 15 ml tubes at 5 ml per tube and stored frozen at −20°C. Positive Hybridomas are fed HCF through the first subcloning and aregradually weaned. It is not necessary to continue to supplement unlessyou have a particularly difficult hybridoma clone. This and otheradditives have been shown to be more effective in promoting newhybridoma growth than conventional feeder layers.

ii) Procedure

To generate monoclonal antibodies, mice are immunized with 5-50 ug ofantigen either intra-peritoneally (i.p.) or by intravenous injection inthe tail vein (i.v.). Typically, the antigen used is a recombinantprotein that is generated as described above. The primary immunizationtakes place 2 months prior to the harvesting of splenocytes from themouse and the immunization is typically boosted by i.v. injection of5-50 ug of antigen every two weeks. At least one week prior to expectedfusion date, a fresh vial of myeloma cells is thawed and cultured.Several flasks at different densities are maintained in order that aculture at the optimum density is ensured at the time of fusion. Theoptimum density is determined to be 3-6×10⁵ cells/ml. Two to five daysbefore the scheduled fusion, a final immunization is administered of ˜5ug of antigen in PBS i.p. or i.v.

Myeloma cells are washed with 30 ml serum free media by centrifugationat 500×g at 4° C. for 5 minutes. Viable cell density is determined inresuspended cells using hemocytometry and vital stains. Cellsresuspended in complete growth medium are stored at 37° C. during thepreparation of splenocytes. Meanwhile, to test aminopterin sensitivity,1×06 myeloma cells are transferred to a 15 ml conical tube andcentrifuged at 500 g at 4° C. for 5 minutes. The resulting pellet isresuspended in 15 ml of HAT media and cells plated at 2 drops/well on a96 well plate.

To prepare splenocytes from immunized mice, the animals are euthanisedand submerged in 70% EtOH. Under sterile conditions, the spleen issurgically removed and placed in 10 ml of RPMI medium supplemented with20% fetal calf serum in a Petri dish. Cells are extricated from thespleen by infusing the organ with medium >50 times using a 21 g syringe.

Cells are harvested and washed by centrifugation (at 500×g at 4° C. for5 minutes) with 30 ml of medium. Cells are resuspended in 10 ml ofmedium and the density of viable cells determined by hemocytometry usingvital stains. The splenocytes are mixed with myeloma cells at a ratio of5:1 (spleen cells: myeloma cells). Both the myeloma and spleen cells arewashed 2 more times with 30 ml of RPMI-CMNS. Spin at 800 rpm for 12minutes.

Supernatant is removed and cells are resuspended in 5 ml of RPMI-CMNSand are pooled to bring the volume to 30 ml and spun down as before. Thecell pellet is broken up by gentle tapping and resuspended in 1 ml ofBMB PEG1500 (prewarmed to 37° C.) added dropwise with 1 cc needle over 1minute.

RPMI-CMNS is added to the PEG cells to slowly to dilute out the PEG.Cells are centrifuged and diluted in 5 ml of Complete media and 95 ml ofClonacell Medium D (HAT) media (with 5 ml of HCF). The cells are platedout at 10 ml per small

Myeloma/HAT control is prepared as follows. Dilute about 1000 P3X63Ag8.653 myeloma cells into 1 ml of medium D and transfer into a singlewell of a 24 well plate. Plates are placed in incubator, with two platesinside of a large petri plate, with an additional petri plate full ofdistilled water, for 10-18 days under 5% CO2 overlay at 37° C. Clonesare picked from semisolid agarose into 96 well plates containing 150-200ul of CM-HT. Supernatants are screened 4 days later in ELISA, andpositive clones are moved up to 24 well plates. Heavy growth willrequire changing of the media at day 8 (+/−150 ml). One should furtherdecrease the HCF to 0.5% (gradually—2%, then 1%, then 0.5%) in thecloning plates.

For further references see Kohler G, and C. Milstein Continuous culturesof fused cells secreting antibody of predefined specificity. 1975.Nature 256: 495-497; Lane, R. D. A short duration polyethylene glycolfusion technique for increasing production of monoclonalantibody-secreting hybridomas. 1985. J. Immunol. Meth. 81: 223-228;

Harlow, E. and D. Lane. Antibodies: A laboratory manual Cold SpringHarbour Laboratory Press. 1988; Kubitz, D. The Scripps ResearchInstitute. La Jolla. Personal Communication; Zhong, G., Berry, J. D.,and Choukri, S. (1996) Mapping epitopes of Chlamydia trachomatisneutralizing monoclonal antibodies using phage random peptide libraries.J. Indust. Microbiol. Biotech. 19, 71-76; Berry, J. D., Licea, A.,Popkov, M., Cortez, X., Fuller, R., Elia, M., Kerwin, L., and C. F.Barbas III. (2003) Rapid monoclonal antibody generation via dendriticcell targeting in vivo. Hybridoma and Hybridomics 22 (1), 23-31.

6. Expression Validation

mRNA Expression Validation by TAQMAN

Expression of mRNA was quantitated by RT-PCR using TaqMan® technology.The Taqman system couples a 5′ fluorogenic nuclease assay with PCR forreal time quantitation. A probe was used to monitor the formation of theamplification product.

Total RNA was isolated from cancer-niodel cell lines using the RNEasykit® (Qiagen) per manufacturer's instructions and included DNasetreatment. Normal human tissue RNAs were acquired from commercialvendors (Ambion, Austin, Tex.; Stratagene, La Jolla, Calif., BioChainInstitute, Newington, N.H.) as were RNAs from matched disease/normaltissues.

Target transcript sequences were identified for the differentiallyexpressed peptides by searching the BlastP database. TaqMan assays (PCRprimer/probe set) specific for those transcripts were identified bysearching the Celera Discovery System™ (CDS) database. The assays weredesigned to span exon-exon borders and do not amplify genomic DNA.

The TaqMan primers and probe sequences were as designed by AppliedBiosystems (AB) as part of the Assays on Demand™ product line or bycustom design through the AB Assays by Design^(SM) service.

RT-PCR was accomplished using AmpliTaqGold and MultiScribe reversetranscriptase in the One Step RT-PCR Master Mix reagent kit (AB)according to the manufacturer's instructions. Probe and primerconcentrations are 250 nM and 900 nM, respectively, in a 15 μl reaction.For each experiment, a master mix of the above components was made andaliquoted into each optical reaction well. Eight nanograms of total RNAwas the template. Each sample was assayed in triplicate. QuantitativeRT-PCR is performed using the ABI Prism® 7900HT Sequence DetectionSystem (SDS). Cycling parameters follow: 48° C. for 30 min. for onecycle; 95° C. for 10 min for one cycle; 95° C. for 15 sec, 60° C. for 1min. for 40 cycles.

The SDS software calculates the threshold cycle (C_(T)) for eachreaction, and C_(T) values were used to quantitate the relative amountof starting template in the reaction. The C_(T) values for each set ofthree reactions were averaged for all subsequent calculations

Data were analyzed for fold differences in expression using anendogenous control for normalization, and measuring expression expressedrelative to a normal tissue or normal cell line reference. The choice ofendogenous control was determined empirically by testing variouscandidates against the cell line and tissue RNA panels and selecting theone with the least variation in expression. Relative changes inexpression were quantitated using the 2^(−ΔΔCT) Method. Livak, K. J. andSchmittgen, T. D. (2001) Methods 25: 402-408; User bulletin #2: ABIPrism: 7700 Sequence Detection System.

Protein Expression Validation by Westen

Western blot analysis of target proteins was carried out using wholecell extracts prepared from each of the pancreatic cell lines. To makecell extracts, the cells were resuspended in Lysis buffer (125 mM Tris,pH 7.5, 150 mM NaCl, 2% SDS, 5 mM EDTA, 0.5% NP-40) and passed through a20-gauge needle. Lysates were centrifuged at 5,000×g for 5 minutes at 4°C. The supernatants were collected and a protease inhibitor cocktail(Sigma) was added. The Pierce BCA assay was used to quantitate totalprotein. Samples were separated by SDS-PAGE and transferred to either anitrocellulose or PVDF membrane. The Western Breeze kit from Invitrogenwas used for western blot analysis. Primary antibodies were eitherpurchased from commercially available sources or prepared using one ofthe methods described in Section 5. For this application, antibodieswere typically diluted 1:500 to 1:10,000 in diluent buffer. Blots weredeveloped using Pierce NBT.

Results

Both mRNA expression and protein expression were performed and manytargets were found to have a positive correlation between mRNA andprotein expression relative to Hs766t. The expression of each of thetargets was first analyzed in the pancreas cell line panel to show thatin fact mRNA expression is also upregulated and that it correlates withthe protein data obtained from the LC/MS. The exemplary targets areMuc4, Kunitz-1 protease inhibitor, Kunitz-2 protease inhibitor, Neutralamino acid transporter B(0), Transmembrane 4 superfamily member 3, KIAA1363, Gs3786, HSPC as well as Tissue Factor.

In addition to Hs766t, mRNAs from normal pancreas tissue or other normaltissues were obtained for control. The targets listed above showed highmRNA expression in pancreatic cancer cell line. Moreover, mRNAs frompancreatic tumors were analysed and were shown to be overexpressed intarget such as TM4SF3.

FIG. 4 shows the Kunitz-type 1 protease inhibitor mRNA expression inpancreatic cell lines. By using Hs766t as a control, the result showsthat Kunitz—type 1 protease inhibitor is overexpressed in AsPc-1,BxPc-3, Capan-2, HPAC, HPAF-II, MPanc-96, Panc1 as well as SU 86.86.Moreover, the data is also consistent with the result from detection ofthe target by using LC/MS with ICAT labeling (see the method below).

FIG. 10 shows that both Gs3786 and Retinol Dehydrogenase Type II homologwere found to be over-expressed in several cell lines relative to thereference cell line Hs766t.

Tissue Flow Cytometry Analysis

Post tissue processing, cells were sorted by flow cytometry known in theart to enrich for epithelial cells. Alternatively, cells isolated frompancratic tissue were stained directly with EpCAM (for epithelial cells)and the specific antibody to PCAT. Cell numbers and viability weredetermined by PI exclusion (GUAVA) for cells isolated from both normaland tumor pancreatic tissue. A minimum of 0.5×10⁶ cells were used foreach analysis. Cells were washed once with Flow Staining Buffer (0.5%BSA, 0.05% NaN3 in D-PBS). To the cells, 20 ul of each antibody for PCATwere added. An additional 5 ul of EpCAM antibody conjugated to APC wereadded when unsorted cells were used in the experiment. Cells wereincubated with antibodies for 30 minutes at 4° C. Cells were washed oncewith Flow Staining Buffer and either analyzed immediately on the LSRflow cytometry apparatus or fixed in 1% formaldehyde and store at 4° C.until LSR analysis. The antibodies used to detect PCAT targets were allpurchased by BD Biosciences and PE-conjugated. The isotype controlantibody used for these experiments was PE-conjugated mouse IgG1k.

Tumors tissue from pancreatic cancer and from Xenograft mouse pancreatictissues were obtained and detected via Flow Cytometry analysis. Tissuefactor was shown to be overexpressed among the pancreatic tumor tissues(data not shown). Xenograft mouse was obtained by using cultured humanBXPC pancreatic cells (ATCC) that were injected subcutaneously in BALBnude mouse. The pancreatic tumors were obtained from the xenograft mousefor further FACS study.

7. Detection and Diagnosis of PCAT by Liquid Chromatography and MassSpectrometry (LC/MS)

The differential expression of proteins in disease and healthy samplesare quantitated using Mass Spectrometry and ICAT (Isotope Coded AffinityTag) labeling, which is known in the art. ICAT is an isotope labeltechnique that allows for discrimination between two populations ofproteins, such as a healthy and a disease sample that are pooledtogether for experimental purposes or two acquisitions of the samesample for classification of true sample peptides from LC/MS noiseartifacts. The LC/MS spectra are collected for the labeled samples andprocessed using the following steps:

The raw scans from the LC/MS instrument are subjected to peak detectionand noise reduction software. Filtered peak lists are then used todetect “features” corresponding to specific peptides from the originalsample(s). Features are characterized by their mass/charge, charge,retention time, isotope pattern and intensity.

Similar experiments can be repeated in order to increase the confidencein detection of a peptide. These multiple acquisitions arecomputationally aggregated into one experiment. Experiments involvinghealthy and disease samples use the known effects of the ICAT label toclassify the peptides as originating from a particular sample or fromboth samples. The intensity of a peptide present in both healthy anddisease samples is used to calculate the differential expression, orrelative abundance, of the peptide. The intensity of a peptide foundexclusively in one sample is used to calculate a theoretical expressionratio for that peptide (singleton). Expression ratios are calculated foreach peptide of each replicate of the experiment (for example, Table 1).

Statistical tests are performed to assess the robustness of the data andselect statistically significant differentials. To assess generalquality of the data, one: a) ensure that similar features are detectedin all replicates of the experiment; b) assess the distribution of thelog ratios of all peptides (a Gaussian was expected); c) calculate theoverall pair wise correlations between ICAT LC/MS maps to ensure thatthe expression ratios for peptides are reproducible across the multiplereplicates; d) aggregate multiple experiments in order to compare theexpression ratio of a peptide in multiple diseases or disease samples.

8. Expression Validation by IHC in Tissue Sections

Tissue Sections

Paraffin embedded, fixed tissue sections were obtained from a panel ofnormal tissues (Adrenal, Bladder, Lymphocytes, Bone Marrow, Breast,Cerebellum, Cerebral cortex, Colon, Endothelium, Eye, Fallopian tube,Small Intestine, Heart, Kidney [glomerulus, tubule], Liver, Lung, Testesand Thyroid) as well as 30 tumor samples with matched normal adjacenttissues from pancreas, lung, colon, prostate, ovarian and breast. Inaddition, other tissues are selected for testing such as bladder renal,hepatocellular, pharyngeal and gastric tumor tissues.

Esophageal replicate sections were also obtained from numerous tumortypes (Bladder Cancer, Lung Cancer, Breast Cancer, Melanoma, ColonCancer, Non-Hodgkins Lymphoma, Endometrial Cancer, Ovarian Cancer, Headand Neck Cancer, Prostate Cancer, Leukemia [ALL and CML] and RectalCancer). Sections were stained with hemotoxylin and eosin andhistologically examined to ensure adequate representation of cell typesin each tissue section.

An identical set of tissues will be obtained from frozen sections andare used in those instances where it is not possible to generateantibodies that are suitable for fixed sections. Frozen tissues do notrequire an antigen retrieval step.

Paraffin Fixed Tissue Sections

Hemotoxylin and Eosin staining of paraffin embedded, fixed tissuesections. Sections were deparaffinized in 3 changes of xylene or xylenesubstitute for 2-5 minutes each. Sections were rinsed in 2 changes ofabsolute alcohol for 1-2 minutes each, in 95% alcohol for 1 minute,followed by 80% alcohol for 1 minute. Slides were washed well in runningwater and stained in Gill solution 3 hemotoxylin for 3 to 5 minutes.Following a vigorous wash in running water for 1 minute, sections werestained in Scott's solution for 2 minutes. Sections were washed for 1min in running water then conterstained in Eosin solution for 2-3minutes depending upon development of desired staining intensity.Following a brief wash in 95% alcohol, sections were dehydrated in threechanges of absolute alcohol for 1 minute each and three changes ofxylene or xylene substitute for 1-2 minutes each. Slides werecoverslipped and stored for analysis.

Optimisation of Antibody Staining

For each antibody, a positive and negative control sample was generatedusing data from the ICAT analysis of the pancreatic cancer cell lines.Cell lines were selected that are known to express low levels of aparticular target as determined from the ICAT data. This cell line wasthe reference normal control “Hs766T”. Similarly, a pancreatic tumourline was selected that was determined to overexpress the target wasselected.

Antigen Retrieval

Sections were deparaffinized and rehydrated by washing 3 times for 5minutes in xylene; two times for 5 minutes in 100% ethanol; two timesfor 5 minutes in 95% ethanol; and once for 5 minutes in 80% ethanol.Sections were then placed in endogenous blocking solution (methanol+2%hydrogen peroxide) and incubated for 20 minutes at room temperature.Sections were rinsed twice for 5 minutes each in deionized water andtwice for 5 minutes in phosphate buffered saline (PBS), pH 7.4.Alternatively, where necessary sections were deparrafinized by HighEnergy Antigen Retrieval as follows: sections were washed three timesfor 5 minutes in xylene; two times for 5 minutes in 100% ethanol; twotimes for 5 minutes in 95% ethanol; and once for 5 minutes in 80%ethanol. Sections were placed in a Coplin jar with dilute antigenretrieval solution (10 mM citrate acid, pH 6). The Coplin jar containingslides was placed in a vessel filled with water and microwaved on highfor 2-3 minutes (700 watt oven). Following cooling for 2-3 minutes,steps 3 and 4 were repeated four times (depending on tissue), followedby cooling for 20 minutes at room temperature. Sections were then rinsedin deionized water, two times for 5 minutes, placed in modifiedendogenous oxidation blocking solution (PBS+2% hydrogen peroxide) andrinsed for 5 minutes in PBS.

Blocking and Staining

Sections were blocked with PBS/1% bovine serum albumin (PBA) for 1 hourat room temperature followed by incubation in normal serum diluted inPBA (2%) for 30 minutes at room temperature to reduce non-specificbinding of antibody. Incubations were performed in a sealed humiditychamber to prevent air-drying of the tissue sections. (The choice ofblocking serum was the same as the species of the biotinylated secondaryantibody). Excess antibody is gently removed by shaking and sectionscovered with primary antibody diluted in PBA and incubated either atroom temperature for 1 hour or overnight at 4° C. (Care was taken thatthe sections do not touch during incubation). Sections were rinsed twicefor 5 minutes in PBS, shaking gently. Excess PBS was removed by gentlyshaking. The sections were covered with diluted biotinylated secondaryantibody in PBA and incubated for 30 minutes to 1 hour at roomtemperature in the humidity chamber. If using a monoclonal primaryantibody, addition of 2% rat serum was used to decrease the backgroundon rat tissue sections. Following incubation, sections were rinsed twicefor 5 minutes in PBS, shaking gently. Excess PBS was removed andsections incubated for 1 hour at room temperature in Vectastain ABCreagent (as per kit instructions). The lid of the humidity chamber wassecured during all incunations to ensure a moist environment. Sectionswere rinsed twice for 5 minutes in PBS, shaking gently.

Develop and Counterstain

Sections were incubated for 2 minutes in peroxidase substrate solutionthat was made up immediately prior to use as follows: 10 mgdiaminobenzidine (DAB) dissolved in 10 ml 50 mM sodium phosphate buffer,pH 7.4; 12.5 microliters 3% CoCl₂/NiCl₂ in deionized water; 1.25microliters hydrogen peroxide

Slides were rinsed well three times for 10 min in deionized water andcounterstained with 0.01% Light Green acidified with 0.01% acetic acidfor 1-2 minutes depending on intensity of counterstain desired.

Slides were rinsed three times for 5 minutes with deionized water anddehydrated two times for 2 minutes in 95% ethanol; two times for 2minutes in 100% ethanol; and two times for 2 minutes in xylene. Stainedslides were mounted for visualization by microscopy.

Results

Tissue Factor: The protein was overexpressed in fourteen out of twentytwo pancreatic tumor and two out seven pancreatic tumor metastases. Innormal tissues, antibody to tissue factor or CD142 showed minimalstaining. Occasional cytoplasmic staining was present in subsets ofinflammatory cells, specifically lymphocytes and mast cells. Gastricchief cells showed cytoplasmic and nuclear staining. Many cell types andtissues, including urothelium, breast epithelium, respiratoryepithelium, adrenal cortex, ovarian stroma, endometrial stroma,pancreatic acinar epithelium, and placental trophoblasts showed focalnuclear staining. However, in contrast to normal tissues, a number ofmalignancies showed significant membranous and cytoplasmic staining inmalignant cells among the tumor tissues. The most prominent positivitywas identified in pancreatic carcinoma, followed by prostatic carcinoma.Individual samples of colon, lung, breast and ovarian carcinoma showedpositive staining. The color scale or number scale indicates the colorintensity of the staining. Each number represents 100% of staining ofall the tumor cells at this staining scale (see FIG. 1). In addition,CD142 (tissue factor) is overexpressed by 2 pathology grades relative tonormal comparative cell type in esophageal 10%, pharyngeal 20%, gastric10%.

Decay Accelerating Factor (DAF: The protein was overexpressed in sevenout of twenty-three pancreatic tumors. The target also overexpressed incolon cancer. DAF was overexpressed by 2 pathology grades in esophageal20%, liver 30%, gastric 30%.

Mucin 4: Mucin 4 is a transmembrane mucin composed of two subunits (aand b) generated from proteolytic cleavage, wherein a is extracellularand b is transmembrane. The b subunit contains two EGF-like domains andinteracts with ErbB2 and the ErbB2/ErbB3 heterodimer with the presenceof neuregulin. Interaction with ErbB2 results phosphorylation of ErbB2and upregulation of cyclin-dependent kinase inhibitor p27kip. Theintereaction causes cell cycle arrest and epithelial celldifferentiation. Interaction of Mucin 4 with ErbB2/B3 results inhyperphosphorylation of the ErbB2/ErbB3/NRG heterotrimer and activationof MAPK, PI3K pathways with downregulation of p27kip. This results inthe stimulation of epithelial cell proliferation. Apoptosis is repressedby expression of Mucin 4 and tumor cell growth therefore stimulated.

In normal tissues, antibody to Mucin 4 showed the most prominentpositive staining in colonic epithelium and scattered individual cellswithin the prostatic epithelium. Additional positive cell types includedpancreatic islets, urothelium, astrocytes, gastric chief cells, andsubsets of renal tubular epithelial cells. Focal positivity was presentin the adrenal cortex, en dometrium, and testis. Rarely, inflammatorycells and single cells (neuroendocrine cells) in the intestinalepithelium were positive. Among malignancies tumor tissues, FIG. 3showed that Mucin 4 protein is overexpressed in breast, non-small lung,prostate, ovarian and pancreatic cancers.

The Mucin 4 was overexpressed in fourteen out of twenty four pancreatictumors and six out of eight metastatic tissues. The protein wasexpressed in several normal epithelia. However, both Mucin 4 and ErbB3were overexpressed in pancreatic tumors. Mucin 4 was also overexpressedin breast cancer. In addition, Muc4 is overexpressed by 2 pathologygrades in esophageal 80% (relative to gastric epithelium), renal cellcarcinoma 90%, hepatocellular 20%, pharyngeal 70% (relative to gastricepithelium), gastric 20%.

ErbB3: The protein was specifically expressed in pancreatic tumor BxPC3cell line and Breast cancer MCF7 cell line by Western analysis. FurtherIHC data showed that ErbB3 is overexpressed in 50% of pancreatic cancer,30% ovarian cancer and 10% non-small cell lung cancer. Overlap ofexpression of Mucin 4 and ErbB3 was predominantly tumor specific whereboth are overexpressed.

Kunitz Inhibitor-1: the protein was overexpressed in thirteen out oftwenty three pancreatic tumors and three out seven metastatic pancreaticcancer tissues. In normal tissues, antibody to Kunitz Inhibitor-1 orHAI-1 showed the most prominent positivity in placental trophoblasts.Colonic epithelium was also positive. Less intense positivity waspresent in urothelium and endometrial glands. Focal positivity waspresent in breast epithelium, respiratory epithelium, centroacinar cellsin the pancreas, subsets of renal tubular epithelial cells, tonsillarepithelium, and hepatic bile ducts. However, in tumor tissues, amajority of samples of all subtypes tested showed positive staining ofmalignant cells. The staining was often membranous and diffused withinthe samples. A few samples of breast and lung cancer contained adjacentbenign epithelium, which showed significantly less intense staining thanthe malignant cells. In pancreatic carcinoma, the level of staining inmalignant cells was frequently similar to that in benign centroacinarcells, but in a few cases, the staining was less intense in malignantcells than in the adjacent benign epithelium. FIG. 2 concludes thatKunitz inhibitor-1 was overexpressed in breast, non-small cell lung,prostate, ovarian as well as pancreatic cancers. In addition, Kunitz-1is overexpressed by 2 pathology grades in: esophageal 40%, renal cellcarcinoma 40%, hepatocellular 25%, pharyngeal 30%, gastric 40%.

Transglutaminase-2 (TGM2): TGM2 protein was overexpressed in seventeenout of twenty four pancreatic tumors and seven out of seven metastatictissues. Pathology study noted overexpression in capilliary endotheliumincluding neovascular capillaries. TGM2 protein was also overexpressedin four out of ten ovarian tumors and overexpressed in non small celllung tumor, prostate tumor. TGM2 was overexpressed by 2 pathology gradesin esophageal 10%, renal 30%, liver 10%, pharyngeal 20%, gastric 10%.

Neutral Amino Acid Transporter (ASCT2): ASCT2 protein was overexpressedin sixteen out of twenty seven pancreatic tumors (60%) and four out ofseven pancreatic metastatic tissues. The protein was also overexpressedin 40% prostate cancer and 30% ovarian cancer tissues. Neutral aminoacid transporter is also overexpressed by 2 pathology grades in:esophageal 10% hepatocellular 75%

CD166: CD166 protein was overexpressed in 10% pancreatic, colon andovarian tumor, in 50% overexpressed in breast tumors.

CD46: CD46 protein was overexpressed in 10% pancreatic tumor and in 30%non-small cell lung tumors.

Kunitz Type inhibitor-2 (HAI-2): HAI-2 was overexpressed in 40%pancreatic tumors and in 10% breast and ovarian tumors.

Na/K ATPase Beta-3: Na/K ATPase Beta-3 was overexpressed in pancreatictumor, ovarian tumor.

GS3786: GS3786 was overexpressed in pancreatic tumorand prostate tumor.

Diminuto-like: Diminuto-like protein is overexpressed (by 2 pathologygrades relative to normal comparative cell type) in 20% pancreatic, 40%prostate, 20% breast and 10% colon tumors

9. IHC Staining of Frozen Tissue Sections

Fresh tissues are embedded carefully in OCT in plastic mold, withouttrapping air bubbles surrounding the tissue. Tissues are frozen bysetting the mold on top of liquid nitrogen until 70-80% of the blockturns white at which point the mold is placed on dry ice. The frozenblocks were stored at −80° C. Blocks are sectioned with a cryostat withcare taken to avoid warming to greater than −10° C. Initially, the blockis equilibrated in the cryostat for about 5 minutes and 6-10 mm sectionsare cut sequentially. Sections are allowed to dry for at least 30minutes at room temperature. Following drying, tissues are stored at 4°C. for short term and −80° C. for longterm storage.

Sections are fixed by immersing in acetone jar for 1-2 minutes at roomtemperature, followed by drying at room temperature. Primary antibody isadded (diluted in 0.05 M Tris-saline [0.05 M Tris, 0.15 M NaCl, pH 7.4],2.5% serum) directly to the sections by covering the section dropwise tocover the tissue entirely. Binding is carried out by incubation achamber for 1 hour at room temperature. Without letting the sections dryout, the secondary antibody (diluted in Tris-saline/2.5% serum) is addedin a similar manner to the primary and incubated as before (at least 45minutes). Following incubation, the sections are washed gently inTris-saline for 3-5 minutes and then in Tris-saline/2.5% serum foranother 3-5 minutes. If a biotinylated primary antibody is used, inplace of the secondary antibody incubation, slides are covered with 100ul of diluted alkaline phosphatase conjugated streptavidin, incubatedfor 30 minutes at room temperature and washed as above. Sections areincubated with alkaline phosphatse substrate (1 mg/ml Fast Violet; 0.2mg/ml Napthol AS-MX phosphate in Tris-Saline pH 8.5) for 10-20 minutesuntil the desired positive staining is achieved at which point thereaction is stopped by washing twice with Tris-saline. Slides arecounter-stained with Mayer's hematoxylin for 30 seconds and washed withtap water for 2-5 minutes. Sections are mounted with Mount coverslipsand mounting media.

10. Assay for Antibody Dependent Cellular Cytotoxicity

Cultured tumor cells are labeled with 100 μCi ⁵¹Cr for 1 hour;Livingston, P. O., Zhang, S., Adluri, S., Yao, T.-J., Graeber, L.,Ragupathi, G., Helling, F., & Fleischer, M. (1997). Cancer Immunol.Immunother. 43, 324-330. After washing three times with culture medium,cells are resuspended at 10⁵/ml, and 100 μl/well are plated onto 96-wellround-bottom plates. A range of antibody concentrations are applied tothe wells, including an isotype control together with donor peripheralblood mononuclear cells that are plated at a 100:1 and 50:1 ratio. Afteran 18-h incubation at 37° C., supernatant (30 μl/well) is harvested andtransferred onto Lumaplate 96 (Packard), dried, and read in a PackardTop-Count NXT γcounter. Each measurement is carried out in triplicate.Spontaneous release is determined by cpm of tumor cells incubated withmedium and maximum release by cpm of tumor cells plus 1% Triton X-100(Sigma). Specific lysis is defined as: % specific lysis=[(experimentalrelease-spontaneous release)/(maximum release-spontaneous release)]×100.The percent ADCC is expressed as peak specific lysis postimmunesubtracted by preimmune percent specific lysis. A doubling of the ADCCto >20% is considered significant.

11. Assay for Complement Dependent Cytotoxicity

Chromium release assays to assess complement-mediated cytotoxicity areperformed for each patient at various time points; Dickler, M. N.,Ragupathi, G., Liu, N. X., Musselli, C., Martino, D. J., Miller, V. A.,Kris, M. G., Brezicka, F. T., Livingston, P. O. & Grant, S.C. (1999)Clin. Cancer Res. 5, 2773-2779. Cultured tumor cells are washed inFCS-free media two times, resuspended in 500 μl of media, and incubatedwith 100 μCi ⁵¹Cr per 10 million cells for 2 h at 37° C. The cells arethen shaken every 15 min for 2 h, washed 3 times in media to achieve aconcentration of approximately 20,000 cells/well, and then plated inround-bottom plates. The plates contain either 50 μl cells plus 50 μlmonoclonal antibody, 50 μl cells plus serum (pre- and posttherapy), or50 μl cells plus mouse serum as a control. The plates are incubated in acold room on a shaker for 45 min. Human complement of a 1:5 dilution(resuspended in 1 ml of ice-cold water and diluted with 3% human serumalbumin) is added to each well at a volume of 100 μl. Control wellsinclude those for maximum release of isotope in 10% Triton X-100 (Sigma)and for spontaneous release in the absence of complement with mediumalone. The plates are incubated for 2 h at 37° C., centrifuged for 3min, and then 100 μl of supernatant is removed for radioactivitycounting. The percentage of specific lysis is calculated as follows: %cytotoxicity=[(experimental release-spontaneous release)/(maximumrelease-spontaneous release)]×100. A doubling of the CDC to >20% isconsidered significant.

12. In vitro Assays in Cell Lines

Lipofectamine was purchased from Invitrogen (Carlsbad, Calif.) andGeneSilencer from Gene Therapy Systems (San Diego, Calif.). SyntheticsiRNA oligonucleotides were from Dharmacon (Lafayette, Colo.), Qiagen(Valencia, Calif.) or Ambion (Austin, Tex.) RNeasy 96 Kit was purchasedfrom Qiagen (Valencia, Calif.). Apop-one homogeneous caspase-3/7 kit andCellTiter 96 AQueous One Solution Cell Proliferation Assay were bothpurchased from Promega (Madison, Wis.). Function blocking antibodieswere purchased from Chemicon (Temecula, Calif.), Biotrend (Cologne,Germany) or Alexis Corporation (San Diego, Calif.). Cell invasion assaykits from purchased from Chemicon (Temecula, Calif.). RiboGreen RNAQuantitation Kit was purchased from Molecular probes (Eugene, Oreg.).

RNAi

RNAi was performed by using Smartpools (Dharmacon), 4—for SilencingsiRNA duplexes (Qiagen) or scrambled negative control siRNA (Ambion).Transient transfections were carried out in triplicate by using eitherLipofectamine 2000 from Invitrogen (Carlsbad, Calif.) or by usingGeneSilencer from Gene Therapy Systems (San Diego, Calif.) in methodsdescribed below. 1 to 4 days after transfections, total RNA was isolatedby using the RNeasy 96 Kit (Qiagen) according to manufacturer'sinstructions and expression of mRNA was quantitated by using TaqMantechnology. Protein expression levels were examined by flow cytometryand apoptosis and proliferation assays were performed daily usingApop-one homogeneous caspase-3/7 kit and CellTiter 96 AQueous OneSolution Cell Proliferation Assay (see protocols below).

i) RNAi Transfections—Lipofectamine 2000

Transient transfections were carried out on sub-confluent pancreaticcancer cell lines as previously described. Elbashir, S. M. et al. (2001)Nature 411: 494-498; Caplen, N. J. et al. (2001) Proc Natl Acad Sci USA98: 9742-9747; Sharp, P. A. (2001) Genes and Development 15: 485-490.Synthetic RNA to gene of interest or scrambled negative control siRNAwas transfected using lipofectamine according to manufacturer'sinstructions. Cells were plated in 96 well plates in antibiotic freemedium. The next day, the transfection reagent and siRNA were preparedfor transfection as follows: Each 0.1-1 ul of lipofectamine 2000 and10-150 mM siRNA were resuspended 25 ul serum-free media and incubated atroom temperature for 5 minutes. After incubation, the diluted siRNA andthe lipofectamine 2000 were combined and incubated for 20 minutes atroom temperature. The cells were then washed and the combinedsiRNA-Lipofectamine 2000 reagent added. After further 4 hoursincubation, 50 ul serum containing medium was added to each well. 1 and4 days after transfection, expression of mRNA was quantitated by RT-PCRusing TaqMan technology and protein expression levels were examined byflow cytometry. Apoptosis and proliferation assays were performed dailyusing Apop-one homogeneous caspase-3/7 kit and CellTiter 96 AQueous OneSolution Cell Proliferation Assay (see protocols below).

ii) RNAi Transfections—GeneSilencer

Transient transfections were carried out on sub-confluent pancreaticcancer cell lines as previously described. Elbashir, S. M. et al. (2001)Nature 411: 494-498; Caplen, N. J. et al. (2001) Proc Natl Acad Sci USA98: 9742-9747; Sharp, P. A. (2001) Genes and Development 15: 485-490.Synthetic RNA to gene of interest or scrambled negative control siRNAwas transfected using GeneSilencer according to manufacturer'sinstructions. Cells were plated in 96 well plates in antibiotic freemedium. The next day, the transfection reagent and the synthetic siRNAwere prepared for transfection as follows: predetermined amount of GeneSilencer was diluted in serum-free media to a final volume of 20 ul perwell. After resuspending 10-150 mM siRNA in 20 ul serum-free media, thereagents were combined and incubated at room temperature for 5-20minutes. After incubation, the siRNA-Gene Silencer reagent was added toeach well and incubated in a 37° C. incubator for 4 hours before anequal volume of serum containing media was added back to the culturedcells. The cells were then incubated for 1 to 4 days before mRNA,protein expression and effects on apoptosis and proliferation wereexamined.

Testing of Functional Blocking Antibodies

Sub-confluent pancreatic cancer cell lines are serum-staved overnight.The next day, serum-containing media is added back to the cells in thepresence of 5-50 ng/ml of function blocking antibodies. After 2 or 5days incubation at 37° C., 5% CO₂, antibody binding is examined by flowcytometry and apoptosis and proliferation are examined by usingprotocols described below.

Apoptosis

Apoptosis assay is performed by using the Apop-one homogeneouscaspase-3/7 kit from Promega. Briefly, the caspase-3/7 substrate isthawed to room temperature and diluted 1:100 with buffer. The dilutedsubstrate is then added 1:1 to cells, control or blank. The plates arethen placed on a plate shaker for 30 minutes to 18 hours at 300-500 rpm.The fluorescence of each well is then measured at using an excitationwavelength of 485+/−20 nm and an emission wavelength of 530+/−25 nm.

Proliferation

Proliferation assay is performed by using the CellTiter 96 AQueous OneSolution Cell Proliferation Assay kit from Promega. 20 ul of CellTiter96 AQueous One Solution is added to 100 ul of culture medium. The platesare then incubated for 1-4 hours at 37° C. in a humidified 5% CO₂incubator. After incubation, the change in absorbance is read at 490 nm.One example of the proliferation assay is MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethanoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)proliferation assay.

Cell Invasion

Cell invasion assay is performed by using the 96 well cell invasionassay kit available from Chemicon. After the cell invasion chamberplates are adjusted to room temperature, 100 ul serum-free media isadded to the interior of the inserts. 1-2 hours later, cell suspensionsof 1×10⁶ cells/ml are prepared. Media is then carefully removed from theinserts and 100 ul of prepared cells are added into the insert +/−0 to50 ng function blocking antibodies. The cells are pre-incubated for 15minutes at 37° C. before 150 ul of media containing 10% FBS is added tothe lower chamber. The cells are then incubated for 48 hours at 37° C.After incubation, the cells from the top side of the insert arediscarded and the invasion chamber plates are then placed on a new96-well feeder tray containing 150 ul of pre-warmed cell detachmentsolution in the wells. The plates are incubated for 30 minutes at 37° C.and are periodically shaken. Lysis buffer/dye sulution (4 ul CyQuantDye/300 ul 4× lysis buffer) is prepared and added to each well ofdissociation buffer/cells on feeder tray. The plates are incubated for15 minutes at room temperature before 150 ul is transferred to a new96-well plate. Fluorescence of invading cells is then read at 480 nmexcitation and 520 nm emission.

Receptor Internalization

For quantification of receptor internalization, ELISA assays areperformed essentially as described by Daunt et al. Daunt, D. A., Hurtz,C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol.Pharmacol. 51, 711-720. The cell lines are plated at 6×10⁵ cells per ina 24-well tissue culture dishes that have previously been coated with0.1 mg/ml poly-L-lysine. The next day, the cells are washed once withPBS and incubated in DMEM at 37° C. for several minutes. Agonist to thecell surface target of interest is then added at a pre-determinedconcentration in prewarmed DMEM to the wells. The cells are thenincubated for various times at 37° C. and reactions are stopped byremoving the media and fixing the cells in 3.7% formaldehyde/TBS for 5min at room temperature. The cells are then washed three times with TBSand nonspecific binding blocked with TBS containing 1% BSA for 45 min atroom temperature. The first antibody is added at a pre-determineddilution in TBS/BSA for 1 hr at room temperature. Three washes with TBSfollowed, and cells are briefly reblocked for 15 min at roomtemperature. Incubation with goat anti-mouse conjugated alkalinephosphatase (Bio-Rad) diluted 1:1000 in TBS/BSA iscarried out for 1 h atroom temperature. The cells are washed three times with TBS and acolorimetric alkaline phosphatase substrate isadded. When the adequatecolor change isreached, 100-μl samples are taken for colorimetricreadings.

mRNA Expression

Expression of mRNA is quantitated by RT-PCR using TaqMan® technology.Total RNA is isolated from cancer model cell lines using the RNEasy 96kit (Qiagen) per manufacturer's instructions and included DNasetreatment. Target transcript sequences are identified for thedifferentially expressed peptides by searching the BlastP database.TaqMan assays (PCR primer/probe set) specific for those transcripts areidentified by searching the Celera Discovery System™ (CDS) database. Theassays are designed to span exon-exon borders and do not amplify genomicDNA. The TaqMan; primers and probe sequences are as designed by AppliedBiosystems (AB) as part of the Assays on Demand™ product line or bycustom design through the AB Assays by Design^(SM) service. RT-PCR isaccomplished using AmpliTaqGold and MultiScribe reverse transcriptase inthe One Step RT-PCR Master Mix reagent kit (AB) according to themanufacturers instructions. Probe and primer concentrations are 900 nMand 250 nM, respectively, in a 25 μl reaction. For each experiment, amaster mix of the above components is made and aliquoted into eachoptical reaction well. 5 ul of total RNA is the template. Each sample isassayed in triplicate. Quantitative RT-PCR is performed using the ABIPrism® 7900HT Sequence Detection System (SDS). Cycling parametersfollow: 48° for 30 min. for one cycle; 95° C. for 10 min for one cycle;95° C. for 15 sec, 60° C. for 1 min. for 40 cycles.

The SDS software calculates the threshold cycle (C_(T)) for eachreaction, and C_(T) values are used to quantitate the relative amount ofstarting template in the reaction. The C_(T) values for each set ofthree reactions are averaged for all subsequent calculations.

Total RNA is quantitated by using RiboGreen RNA Quantitation Kitaccording to manufacturer's instructions and the % mRNA expression iscalculated using total RNA for normalization. % knockdown is thencalculated relative to the no addition control.

Results

The assay was performed on analysis of Mucin 4 knowdown. Mucin 4 siRNAinduced apoptosis of ASPC-1 cells 3 days after transfection, and levelof apoptosis was dose-dependent on muscin siRNA (n=3). The level ofapoptosis on day 3 was similar to that given by TRAIL+ actinomycin Dpositive control (FIG. 5). Moreover, FIG. 6 also shows that Mucin 4siRNA inhibited the proliferation of ASPC-1 cells (n=3). FIG. 7 showsthat the level of inhibition was dose-dependent on the concentration ofMucin 4 siRNA (n=3). The result shows that Mucin 4 siRNA can inhibiteabout 50% of viable ASPC-1 cells 3 days after transfection (n=2). BothSMARTPool Mucin 4 siRNA and Mucin 4 duplexl siRNA can induce aninhibition of proliferation and an increase in apoptosis 3 days aftertransfection (FIG. 8). Additional assays were also performed on othertargets: Apoptosis occurred when Na⁺/K⁺ transporting ATPase beta-3 chain(PA-088), GS3786(PA-078), CD49c(PA-009), siRNA were administrated inCacaspase 3/7 analysis in ASPC-1 cell line. Apoptosis occurred whenNa⁺/K⁺ transporting ATPase beta-3 chain, GS3786, and Celera protein RNAiwere administrated in Cacaspase 3/7 analysis in BXPC-3 cell line.Specifically, Na⁺/K⁺ transporting ATPase beta-3 chain increasedapoptosis of ASPC-1 pancreatic cancer cells in a dose-dependent mannerand can decrease proliferation of ASPC-1 pancreatic cancer cells. GS3786siRNA increased apoptosis of ASPC-1 pancreatic cancer cells in adose-dependent manner. It increased apoptosis of BXPC-3 pancreaticcancer cells and decreased proliferation of ASPC-1 and BXPC-3 pancreaticcancer cells. CD49c siRNA increased apoptosis of BXPC-3 pancreaticcancer cells in a dose-dependent manner and decreased proliferation ofBXPC-3 pancreatic cancer cells in a dose-dependent manner. (FIG. 11 andFIG. 12]

13. In Vivo Studies by Using Antibodies

Treatment of Pancreatic Cancer Cells With Monoclonal Antibodies.

Pancreatic cancer cells are seeded at a density of 4×10⁴ cells per wellin 96-well microtiter plates and allowed to adhere for 2 hours. Thecells are then treated with different concentrations of anti-PCATmonoclonal antibody (Mab) or irrelevant isotype matched (anti-rHuIFN-.gamma. Mab) at 0.05, 0.5 or 5.0 mug/ml. After a 72 hour incubation, thecell monolayers are stained with crystal violet dye for determination ofrelative percent viability (RPV) compared to control (untreated) cells.Each treatment group consists of replicates. Cell growth inhibition ismonitored.

Treatment of NIH 3T3 Cells Overexpression PCAT Protein with MonoclonalAntibodies.

NIH 3T3 expressing PCAT protein are treated with differentconcentrations of anti-PCAT MAbs. Cell growth inhibition is monitored.

In Vivo Treatment of NIH 3T3 Cells Overexpressing PCAT with Anti-PCATMonoclonal Antibodies.

NIH 3T3 cells transfected with etiher a PCAT expression plasmidor theneo-DHFR vector are injected into nu/lnu (athymic) mice subcutaneouslyat a dose of 10⁶ cells in 0.1 ml of phosphate-buffered saline. On days0, 1, 5 and every 4 days thereafter, 100 mug (0.1 ml in PBS) of eitheran irrelevant or anti-PCAT monoclonal antibody of the IG2A subclass isinjected intraperitoneally. Tumor occurrence and size are monitored for1 month period of treatment.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention, which are obvious to those skilled inthe field of molecular biology or related fields, are intended to bewithin the scope of the following claims.

1. A method of diagnosing pancreatic cancer in an individual, the methodcomprising detecting a higher level of ASCT2 protein in a sample fromthe individual as compared to levels of ASCT2 protein in pancreatictissues of healthy individuals, wherein the amino acid sequence of theASCT2 protein comprises SEQ ID NO:56, and wherein the higher level ofASCT2 protein indicates that said individual has pancreatic cancer. 2.The method of claim 1, wherein the ASCT2 protein is detected bycontacting the sample with an isolated antibody that selectively bindsto the ASCT2 protein and determining the extent or binding of theantibody to the ASCT2 protein.
 3. The method of claim 2, wherein theantibody is coupled to a detectable substance.